Genes involved in estrogen metabolism

ABSTRACT

The invention concerns genes that have been identified as being involved in estrogen metabolism, and are useful as diagnostic, prognostic and/or predictive markers in cancer. In particular, the invention concerns genes the tumor expression levels of which are useful in the diagnosis of cancers associated with estrogen metabolism, and/or in the prognosis of clinical outcome and/or prediction of drug response of such cancers.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a non-provisional application filed under 37 C.F.R. §1.53(b),claiming priority under 37 C.F.R. §119(e) to U.S. Provisional PatentApplication Ser. No. 60/787,926, filed on Mar. 31, 2006 and to U.S.Provisional Patent Application Ser. No. 60/789,187, filed on Apr. 3,2006, the entire disclosures of which are hereby expressly incorporatedby reference.

FIELD OF THE INVENTION

The present invention concerns genes that have been identified as beinginvolved in estrogen metabolism, and are useful as diagnostic,prognostic and/or predictive markers in cancer. In particular, thepresent invention concerns genes the tumor expression levels of whichare useful in the diagnosis of cancers associated with estrogenmetabolism, and/or in the prognosis of clinical outcome and/orprediction of drug response of such cancers.

DESCRIPTION OF THE RELATED ART

Gene Expression Studies

Oncologists regularly confront treatment decisions regarding whether acancer patient should receive treatment and, if so, what treatment tochoose. These oncologists typically have a number of treatment optionsavailable to them, including different combinations of chemotherapeuticdrugs that are characterized as “standard of care.” Because these“standard of care” chemotherapeutic drugs such as cyclophosphamide,methotrexate, 5-fluorouracil, anthracyclines, taxanes, have limitedefficacy and a spectrum of often severe side effects, it is important toidentify those patients having the highest likelihood of a positiveclinical outcome without chemotherapy (patients with good prognosis) inorder to minimize unnecessary exposure of these patients to the toxicside effects of the chemotherapeutic agents.

For those patients with a poor prognosis it is then important to predictthe likelihood of beneficial response in individual patients toparticular chemotherapeutic drug regimens. Identification of thosepatients most likely to benefit from each available treatment willenhance the utility of “standard of care” treatments, and facilitate thedevelopment of further, more personalized treatment options, includingthe use of already approved drugs that had previously not beenrecommended for the treatment of a particular cancer. The identificationof patients who are more likely or less likely to need and respond toavailable drugs thus could increase the net benefit these drugs have tooffer and decrease net morbidity and toxicity, via more intelligentpatient selection.

Most diagnostic tests currently used in clinical practice are singleanalyte, and therefore do not capture the potential value of knowingrelationships between dozens of different markers. Moreover, diagnostictests are often based on immunohistochemistry, which is notquantitative. Immunohistochemistry often yields different results indifferent laboratories, in part because the reagents are notstandardized, and in part because the interpretations are subjective.RNA-based tests, while potentially highly quantitative, have not beenused because of the perception that RNA is destroyed in tumor specimensas routinely prepared, namely fixed in formalin and embedded in paraffin(FPE), and because it is inconvenient to obtain and store fresh tissuesamples from patients for analysis.

Over the last two decades molecular biology and biochemistry haverevealed hundreds of genes whose activities influence the behavior oftumor cells, their state of differentiation, and their sensitivity orresistance to certain therapeutic drugs. However, with a few exceptions,the status of these genes has not been exploited for the purpose ofroutinely making clinical decisions about drug treatments. In the lastfew years, several groups have published studies concerning theclassification of various cancer types by microarray gene expressionanalysis of thousands of genes (see, e.g. Golub et al., Science286:531-537 (1999); Bhattacharjae et al., Proc. Natl. Acad. Sci. USA98:13790-13795 (2001); Chen-Hsiang et al., Bioinformatics 17 (Suppl.1):S316-S322 (2001); Ramaswamy et al., Proc. Natl. Acad. Sci. USA98:15149-15154 (2001); Martin et al., Cancer Res. 60:2232-2238 (2000);West et al., Proc. Natl. Acad. Sci. USA 98:11462-114 (2001); Sorlie etal., Proc. Natl. Acad. Sci. USA 98:10869-10874 (2001); Yan et al.,Cancer Res. 61:83.75-8380 (2001)). However, these studies have not yetyielded tests routinely used in clinical practice, in large part becausemicroarrays require fresh or frozen tissue RNA and such specimens arenot present in sufficient quantity to permit clinical validation ofidentified molecular signatures.

In the past three years, it has become possible to profile geneexpression of hundreds of genes in formalin-fixed paraffin-embedded(FPE) tissue using RT-PCR technology. Methods have been described thatare highly sensitive, precise, and reproducible (Cronin et al., Am. J.Pathol. 164:35-42 (2004); PCT Publication No. WO 2003/078,662; WO2004/071,572; WO 2004/074,518; WO 2004/065,583; WO 2004/111,273; WO2004/111,603; WO 20051008,213; WO 2005/040,396; WO 2005/039,382; WO2005/064,019, the entire disclosures of which are hereby expresslyincorporated by reference). Because thousands of archived FPE clinicaltissue specimens exist with associated clinical records, such assurvival, drug treatment history, etc., the ability to nowquantitatively assay gene expression in this type of tissue enablesrapid clinical studies relating expression of certain genes to patientprognosis and likelihood of response to treatments. Using data generatedby past clinical studies allows for rapid results because the clinicalevents are historical. In contrast, for example, if one wished to carryout a survival study on newly recruited cancer patients one wouldgenerally need to wait for many years for statistically sufficientnumbers of deaths to have occurred.

Breast Cancer Prognosis and Prediction

Breast cancer is the most common type of cancer among women in theUnited States, and is the leading cause of cancer deaths among womenbetween the ages of 40 and 59.

Because current tests for prognosis and for prediction of chemotherapyresponse are inadequate, breast cancer treatment strategies vary betweenoncologists (Schott and Hayes, J. Clin. Oncol. PMID 15505274 (2004);Hayes, Breast 12;543-9 (2003)). The etiology of certain types of humanbreast cancer involves certain steroid hormones, called estrogens.Estrogens are believed to cause proliferation of breast epithelial cellsprimarily via binding of hormones to estrogen receptors, resulting inmodification of the cellular transcription program. For these reasons,one of the most commonly used markers in selecting a treatment optionfor breast cancer patients is the estrogen receptor 1 (ESR1). Estrogenreceptor-positive (ESR1+) tumors are generally less aggressive thanestrogen receptor negative (ESR1−) tumors, and can often be successfullytreated with anti-estrogens such as tamoxifen (TAM). Conversely, ESR1−tumors are typically more aggressive and are resistant to anti-estrogentreatment. Thus, aggressive chemotherapy is often provided to patientsfor ESR1− tumors. Based on this simple understanding, assays for ESR1levels by immunohistochemistry are currently utilized as one parameterfor making treatment decisions in breast cancer. Generally, lymph nodenegative patients whose tumors are found to be ESR1 positive are treatedwith an anti-estrogen drug, such as tamoxifen (TAM), and patients whosetumors are found to be ESR1 negative are treated with chemotherapy.However, often because of the uncertainty in the currently useddiagnostic procedures, ESR1 positive patients are also prescribedchemotherapy in addition to anti-estrogen therapy, accepting the toxicside effects of chemotherapy in order to modestly decrease the risk ofcancer recurrence. Toxicities include, neuropathy, nausea and othergastrointestinal symptoms, hair loss and cognitive impairment.Recurrence is to be feared because recurrent breast cancer is usuallymetastatic and poorly responsive to treatment.

The human GSTM (GSM) gene family consists of five different closelyrelated isotypes, GSTM1-GSTM5. GSTM proteins conjugate glutathione tovarious electrophilic small molecules, facilitating clearance of theelectrophiles from cells. Evidence exists that several metabolites ofestrogen, including estrogen semi-quinones and estrogen quinones(catechol estrogens), are toxic and mutagenic (Cavalieri et al., ProcNatl Acad Sci 94:10937-42,1997). The activity of one or more GSTMenzymes may limit mutational damage caused by these estrogenmetabolites.

We have reported five independent clinical studies in which GSTM geneexpression was examined by quantitative RT-PCR in formalin-fixed,paraffin embedded primary breast cancer tissues. GSTM expressioncorrelated strongly with favorable clinical outcome in each of thesestudies (Esteban et al., Prog. Proc Am Soc. Clin. Oncol. 22:850abstract, 2003; Cobleigh et al., Clin. Cancer Res (in press); Paik etal., Breast Cancer Res. Treat. 82:A16 abstract, 2003; Habel et al,Breast. Cancer Res. Treat. 88:3019 abstract, 2004: Paik et al, N Engl JMed 351:2817-26, 2004).

In these studies the probe used could not discriminate between GSTM1 andseveral other GSTM family members as a result of the strong sequencesimilarity of the GSTM genes, amplicon size limitations and thestringent sequence criteria for probe-primer design, leaving thepossibility that several of the GSTM genes may be favorable markers.

Clearly, a need exists to identify those patients who are at substantialrisk of cancer recurrence (i.e., to provide prognostic information)and/or likely to respond to chemotherapy (i.e., to provide predictiveinformation). Likewise, a need exists to identify those patients who donot have a significant risk of recurrence, and/or who are unlikely torespond to chemotherapy, as these patients should be spared needlessexposure to these toxic drugs.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the recognitionthat since estrogens may contribute to tumorigenesis and tumorprogression via pathways that are ESR1 independent, treatment decisionsbased primarily or solely on the ESR1 status of a patient areunsatisfactory.

One aspect of the invention is directed to a method of predictingclinical outcome for a subject diagnosed with cancer, comprisingdetermining evidence of the expression level of one or more predictiveRNA transcripts listed in Table 8, or their expression products, in abiological sample comprising cancer cells obtained from said subject,wherein evidence of increased expression of one or more of the geneslisted in Table 8, or the corresponding expression product, indicates adecreased likelihood of a positive clinical outcome. In one embodimentthe subject is a human patient. In one embodiment the expression levelis obtained by a method of gene expression profiling. In one embodimentthe method of gene expression profiling is a PCR-based method. In oneembodiment the expression levels are normalized relative to theexpression levels of one or more reference genes, or their expressionproducts. In one embodiment the clinical outcome is expressed in termsof Recurrence-Free Interval (RFI), Overall Survival (OS), Disease-FreeSurvival (DFS), or Distant Recurrence-Free Interval (DRFI). In oneembodiment the cancer is selected from the group consisting of breastcancer or ovarian cancer. In one embodiment the cancer is breast cancer.

In one embodiment, the method of predicting clinical outcome for asubject diagnosed with cancer comprises determining evidence of theexpression level of at least two of said genes, or their expressionproducts. In another embodiment, the expression levels of at least threeof said genes, or their expression products are determined. In yetanother embodiment, the expression levels of at least four of saidgenes, or their expression products are determined. In a furtherembodiment, the expression levels of at least five of said genes, ortheir expression products are determined.

The method may further comprise the step of creating a reportsummarizing said prediction.

Another aspect of the invention is a method of predicting the durationof Recurrence-Free Interval (RFI) in a subject diagnosed with breastcancer, comprising determining the expression level of one or morepredictive RNA transcripts listed in Table 8 or their expressionproducts, in a biological sample comprising cancer cells obtained fromsaid subject, wherein evidence of increased expression of one or more ofthe genes listed in Table 8, or the corresponding expression product,indicates that said RFI is predicted to be shorter. In one embodimentthe subject is a human patient. In another aspect the expression levelis obtained by a method of gene expression profiling. In one embodimentthe method of gene expression profiling is a PCR-based method. In oneembodiment the expression levels are normalized relative to theexpression levels of one or more reference genes, or their expressionproducts. In one embodiment the clinical outcome is expressed in termsof Recurrence-Free Interval (RFI), Overall Survival (OS), Disease-FreeSurvival (DFS), or Distant Recurrence-Free Interval (DRFI). In oneembodiment the cancer is selected from the group consisting of breastcancer or ovarian cancer. In one embodiment the cancer is breast cancer.

One aspect of the method of predicting the duration of Recurrence-FreeInterval (RFI), for a subject diagnosed with cancer, comprisesdetermining evidence of the expression level of at least two of saidgenes, or their expression products. In one embodiment the expressionlevels of at least three of said genes, or their expression products aredetermined. In another embodiment the expression levels of at least fourof said genes, or their expression products are determined. In anotherembodiment the expression levels of at least five of said genes, ortheir expression products are determined.

One aspect of the methods of this invention is that if the RFI ispredicted to be shorter, said patient is subjected to further therapyfollowing surgical removal of the cancer. In one aspect, the therapy ischemotherapy and/or radiation therapy.

One aspect of the methods of this invention is that the expression levelof one or more predictive RNA transcripts or their expression productsof one or more genes selected from the group consisting of CAT, CRYZ,CYP4Z1, CYP17A1, GPX1, GPX2, GSTM1, GSTM2, GSTM3, GSTM4, GSTM5, GSTP1,NQO1, PRDX3, and SC5DL is determined.

One aspect of the methods of this invention is that the expression levelof one or more predictive RNA transcripts or their expression productsof one or more genes selected from the group consisting of GSTM1, GSTM2,GSTM3, GSTM4, GSTM5 and GSTP1 is determined.

One aspect of the methods of this invention is that the expression levelof one or more predictive RNA transcripts or their expression productsof one or more genes selected from the group consisting of GSTM2 andGSTM4 is determined.

One aspect of the methods of this invention is that the expression levelof one or more predictive RNA transcripts or their expression productsof one or more genes selected from the group consisting of GSTM1 andGSTM3 is determined.

One aspect of the methods of this invention is that the expression levelof one or more predictive RNA transcripts or their expression productsof one or more genes selected from the group consisting of CAT, PRDX3,GPX1, and GPX2 is determined.

One aspect of the methods of this invention is that the expression levelof one or more predictive RNA transcripts or their expression productsof one or more genes selected from the group consisting of PRDX3, GPX1and GPX2 is determined.

One aspect of the methods of this invention is that the expression levelof one or more predictive RNA transcripts or their expression productsof one or more genes selected from the group consisting of GPX1 and GPX2is determined.

One aspect of the methods of this invention is that the expression levelof one or more predictive RNA transcripts or their expression productsof one or more genes selected from the group consisting of CRYZ and NQO1is determined.

One aspect of the methods of this invention is that the expression levelof one or more predictive RNA transcripts or their expression productsof CYP17A1 is determined.

One aspect of the methods of this invention is that the expression levelof one or more predictive RNA transcripts or their expression productsof one or more genes selected from the group consisting of SC5DL andCYP4Z1 is determined.

In another aspect, this invention concerns a method for preparing apersonalized genomics profile for a patient comprising the steps of

-   -   (a) subjecting RNA extracted from a tissue obtained from the        patient to gene expression analysis;    -   (b) determining the expression level in the tissue of one or        more genes selected from the gene set listed in Table 8 wherein        the expression level is normalized against a control gene or        genes and optionally is compared to the amount found in a cancer        reference set and    -   (c) creating a report summarizing the data obtained by said gene        expression analysis.

Another embodiment of this invention is a method for amplification of agene listed in Table 8 by polymerase chain reaction (PCR) comprisingperforming said per by using amplicons listed in Table 7 and aprimer-probe set listed in Table 6.

Another embodiment of this invention is a PCR primer-probe set listed inTable 6.

Another embodiment of this invention is a PCR amplicon listed in Table7.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

FIG. 1 shows the sequence alignment of the GSTM1 and GSTM2 ampliconswith the corresponding regions of other GSTM family members.

FIG. 2 shows the distribution of RT-PCR signals as CT values (X-axis)across the 125 breast cancer patients (Y-axis) for GSTM1.1, GSTM1int5.2and GSTM2int4.2.

FIG. 3 shows the distribution of RT-PCR signals as CT values for 22human subjects for the different GSTM amplicons.

FIG. 4 shows the similarity and chromosome location of the GSTM genes.

FIG. 5 shows the cellular pathways which are the possible basis for thecorrelation of GSTM expression with good outcome.

FIG. 6 shows specific pathways for the degradation, modification andclearance of key estrogens, estrone and estradiol.

FIG. 7 shows specific pathways for the synthesis of key estrogens,estrone and estradiol, from cholesterol.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT A. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs, Singleton et al., Dictionary ofMicrobiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,N.Y. 1994); and Webster's New World™ Medical Dictionary, 2nd Edition,Wiley Publishing Inc., 2003, provide one skilled in the aft with ageneral guide to many of the terms used in the present application. Forpurposes of the present invention, the following terms are definedbelow.

The term RT-PCR has been variously used in the art to meanreverse-transcription PCR (which refers to the use of PCR to amplifymRNA by first converting mRNA to double stranded cDNA) or real-time PCR(which refers to ongoing monitoring in ‘real-time’ of the amount of PCRproduct in order to quantify the amount of PCR target sequence initiallypresent. The term “RT-PCR’ means reverse transcription PCR. The termquantitative RT-PCR (qRT-PCR) means real-time PCR applied to determinethe amount of mRNA initially present in a sample.

The term “clinical outcome” means any measure of patient statusincluding those measures ordinarily used in the art, such as diseaserecurrence, tumor metastasis, overall survival, progression-freesurvival, recurrence-free survival, and distant recurrence-freesurvival. Distant recurrence-free survival (DRFS) refers to the time (inyears) from surgery to the first distant recurrence.

The term “microarray” refers to an ordered arrangement of hybridizablearray elements, preferably polynucleotide probes, on a substrate.

The term “polynucleotide,” when used in singular or plural, generallyrefers to any polyribonucleotide or polydeoxribonucleotide, which may beunmodified RNA or DNA or modified RNA or DNA. Thus, for instance,polynucleotides as defined herein include, without limitation, single-and double-stranded DNA, DNA including single- and double-strandedregions, single- and double-stranded RNA, and RNA including single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or includesingle- and double-stranded regions. In addition, the term“polynucleotide” as used herein refers to triple-stranded regionscomprising RNA or DNA or both RNA and DNA. The strands in such regionsmay be from the same molecule or from different molecules. The regionsmay include all of one or more of the molecules, but more typicallyinvolve only a region of some of the molecules. One of the molecules ofa triple-helical region often is an oligonucleotide. The term“polynucleotide” specifically includes cDNAs. The term includes DNAs(including cDNAs) and RNAs that contain one or more modified bases.Thus, DNAs or RNAs with backbones modified for stability or for otherreasons are “polynucleotides” as that term is intended herein. Moreover,DNAs RNAs comprising unusual bases, such as inosine, or modified bases,such as tritiated bases, are included within the term “polynucleotides”as defined herein. In general, the term “polynucleotide” embraces allchemically, enzymatically and/or metabolically modified forms ofunmodified polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including simple and complex cells.

The term “oligonucleotide” refers to a relatively short polynucleotide,including, without limitation, single-stranded deoxyribonucleotides,single- or double-stranded ribonucleotides, RNA:DNA hybrids anddouble-stranded DNAs. Oligonucleotides, such as single-stranded DNAprobe oligonucleotides, are often synthesized by chemical methods, forexample using automated oligonucleotide synthesizers that arecommercially available. However, oligonucleotides can be made by avariety of other methods, including in vitro recombinant DNA-mediatedtechniques and by expression of DNAs in cells and organisms.

The term “gene expression” describes the conversion of the DNA genesequence information into transcribed RNA (the initial unspliced RNAtranscript or the mature mRNA) or the encoded protein product. Geneexpression can be monitored by measuring the levels of either the entireRNA or protein products of the gene or subsequences.

The phrase “gene amplification” refers to a process by which multiplecopies of a gene or gene fragment are formed in a particular cell orcell line. The duplicated region (a stretch of amplified DNA) is oftenreferred to as “amplicon.” Often, the amount of the messenger RNA (mRNA)produced, i.e., the level of gene expression, also increases in theproportion of the number of copies made of the particular geneexpressed.

Prognostic factors are those variables related to the natural history ofbreast cancer, which influence the recurrence rates and outcome ofpatients once they have developed breast cancer. Clinical parametersthat have been associated with a worse prognosis include, for example,lymph node involvement, increasing tumor size, and high grade tumors.Prognostic factors are frequently used to categorize patients intosubgroups with different baseline relapse risks. In contrast, treatmentpredictive factors are variables related to the likelihood of anindividual patient's beneficial response to a treatment, such asanti-estrogen or chemotherapy, independent of prognosis.

The term “prognosis” is used herein to refer to the likelihood ofcancer-attributable death or cancer progression, including recurrenceand metastatic spread of a neoplastic disease, such as breast cancer,during the natural history of the disease. Prognostic factors are thosevariables related to the natural history of a neoplastic diseases, suchas breast cancer, which influence the recurrence rates and diseaseoutcome once the patient developed the neoplastic disease, such asbreast cancer. In this context, “natural outcome” means outcome in theabsence of further treatment. For example, in the case of breast cancer,“natural outcome” means outcome following surgical resection of thetumor, in the absence of further treatment (such as, chemotherapy orradiation treatment). Prognostic factors are frequently used tocategorize patients into subgroups with different baseline risks, suchas baseline relapse risks.

The term “prediction” is used herein to refer to the likelihood that apatient will respond either favorably or unfavorably to a drug or set ofdrugs, and also the extent of those responses. Thus, treatmentpredictive factors are those variables related to the response of anindividual patient to a specific treatment, independent of prognosis.The predictive methods of the present invention can be used clinicallyto make treatment decisions by choosing the most appropriate treatmentmodalities for any particular patient. The predictive methods of thepresent invention are valuable tools in predicting if a patient islikely to respond favorably to a treatment regimen, such asanti-estrogen therapy, such as TAM treatment alone or in combinationwith chemotherapy and/or radiation therapy.

The term “long-term” survival is used herein to refer to survival for atleast 3 years, more preferably for at least 8 years, most preferably forat least 10 years following surgery mother treatment.

The term “tumor,” as used herein, refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, breast cancer, ovarian cancer, colon cancer, lung cancer, prostatecancer, hepato cellular cancer, gastric cancer, pancreatic cancer,cervical cancer, liver cancer, bladder cancer, cancer of the urinarytract, thyroid cancer, renal cancer, carcinoma, melanoma, and braincancer.

The “pathology” of cancer includes all phenomena that compromise thewell-being of the patient. This includes, without limitation, abnormalor uncontrollable cell growth, metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, neoplasia, premalignancy,malignancy, invasion of surrounding or distant tissues or organs, suchas lymph nodes, etc.

In the context of the present invention, reference to “at least one,”“at least two,” “at least three,” “at least four,” “at least five,” etc.of the genes listed in any particular gene set means any one or any andall combinations of the genes listed.

The term “node negative” cancer, such as “node negative” breast cancer,is used herein to refer to cancer that has not spread to the lymphnodes.

The terms “splicing” and “RNA splicing” are used interchangeably andrefer to RNA processing that removes introns and joins exons to producemature mRNA with continuous coding sequence that moves into thecytoplasm of an eukaryotic cell.

In theory, the term “exon” refers to any segment of an interrupted genethat is represented in the mature RNA product (B, Lewin. Genes IV CellPress, Cambridge Mass. 1990). In theory the term “intron” refers to anysegment of DNA that is transcribed but removed from within thetranscript by splicing together the exons on either side of it.Operationally, exon sequences occur in the mRNA sequence of a gene asdefined by Ref. SEQ ID numbers. Operationally, intron sequences are theintervening sequences within the genomic DNA of a gene, bracketed byexon sequences and having GT and AG splice consensus sequences at their5′ and 3′ boundaries.

B. Detailed Description

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, and biochemistry,which are within the skill of the art. Such techniques are explainedfully in the literature, such as, “Molecular Cloning: A LaboratoryManual”, 2^(nd) edition (Sambrook et al., 1989); “OligonucleotideSynthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I.Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.);“Handbook of Experimental Immunology”, 4^(th) edition (D. M. Weir & C.C. Blackwell, eds., Blackwell Science Inc., 1987); “Gene TransferVectors for Mammalian Cells” (J. M. Miller & M. P. Calos, eds., 1987);“Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds.,1987); and “PCR: The Polymerase Chain Reaction”, (Mullis et al., eds.,1994). The practice of the present invention will also employ, unlessotherwise indicated, conventional techniques of statistical analyis suchas the Cox Proportional Hazards model (see, e.g. Cox, D. R., and Oakes,D. (1984), Analysis of Survival Data, Chapman and Hall, London, N.Y.).Such techniques are explained fully in the literature.

B.1. General Description of the Invention

As discussed before, the present invention is based, at least in part,on the recognition that since estrogens may contribute to tumorigenesisand tumor progression via pathways that are ESR1 independent, treatmentdecisions based primarily or solely on the ESR1 status of a patient areunsatisfactory.

Estrogen Metabolism

It is known that certain pathways of estrogen degradation involve theproduction of electrophilic estrogen metabolites as well as reactiveoxygen species (ROS), both of which have the potential to damagecellular DNA and thus contribute to carcinogenesis (Cavalieri et al.,Cell. Mol. Life Sci. 59: 665-81 (2002); Thompson and Ambrosone, J. Natl.Cancer Inst. 27: 125-34 (2000)).

The present invention is based on the identification of genes that arebelieved to be involved in the metabolism and/or clearance Of estrogen,and thus in the control of intracellular concentration of electrophilicestrogen metabolites. In a specific embodiment, gene specific probeprimer sets were designed based on the exon and introns sequences of thegenes identified. These probe primer sets may be used in conjunctionwith a variety of clinical samples to identify particular genes withinthe estrogen metabolism group which are prognostic of outcome in aparticular type of cancer and/or have predictive value in determiningpatient response to a particular treatment modality.

Estrogens, including the principle active hormones, estrone andestradiol, can be converted to catechol estrogens (CE) via either2-hydroxylation by cytochrome P4501A1 (CYP1A1) or via 4-hydroxylation bycytochrome P4501B1 (CYP1B1). These catechol estrogens (CE) can befurther metabolized to CE semiquinones and then to CE quinones, whichcompounds are electrophiles and are proven or potential mutagens.(Mitrunen and Hirvonen, Mutation Research, 544: 9-41 (2003); Lieher,Endocrine Reviews, 21:40-54 (2000)). Furthermore, concomitant with theconversion of estrogen semiquinones to estrogen quinones, molecularoxygen is converted to highly reactive superoxide anion, which also candamage DNA.

The presence of electrophilic estrogen metabolites and reactive oxygenspecies could cause mutations in normal cells over time, resulting intumorigenesis and could further cause new mutations in existing tumorcells that may be already compromised in their ability to repair damageto their DNA. The resulting increased burden of mutations could resultin emergence of more aggressive clones in the tumor, more tumoraneuploidy and heterogeneity, with negative consequences for the healthof the patient. Cellular metabolic strategies that would minimize theformation of mutagenic estrogen metabolites or increase the efficiencyof their removal via conversion or clearance would then minimizemutagenic effects and result in more favorable prognosis.

Although a number of studies have been carried out to determine theeffect on breast cancer predisposition risk of allelic variation inestrogen metabolizing genes, little has been done regarding thepotential effect on cancer predisposition or prognosis, of expressionlevels of the various genes that affect cellular levels of mutatgenicestrogen metabolites.

One alternative to the catechol/quinone pathway discussed above is theconversion, by the enzyme cathecol-O-methyl transferase (COMT), ofestrogen catechols to 2-methoxy and 4-methoxy estrogens, compounds thatare much less reactive than the quinones and more readily cleared fromthe cell.

Mutagenic catechol estrogen quinones can be converted back to catecholestrogens through the action of a NADPH-dependent quinone reductase(CRYZ), making them re-available for metabolism via COMT.

Direct clearance of both CE semiquinones and CE quinones can beinitiated by conjugation of the metabolites with glutathione catalyzedby glutathione-S-transferase (GST) enzymes. The GST protein familyincludes GST mu enzymes (GSTM1, GSTM2, GSTM3, GSTM4 and GSTM5), GST pienzyme GSTP1 and GST theta enzyme GSTT1. In addition to the aboveenzymes, membrane-associated glutathione-S-transferase enzymes thatcatalyze the conjugation of glutathione to electrophiles; includingMGST1 and MGST3, have been identified. Membrane-associatedglutathione-S-transferase may also catalyze the reduction of lipidhydroperoxides (see below).

Glutathione, required by GST enzymes, is a tripeptide synthesized fromamino acids in a process the rate-limiting step of which is catalyzed bygamma-glutamylcysteine synthetase, an enzyme composed of a catalyticsubunit (GCLC) and a regulatory subunit (GCLM) that are encoded byseparate genes.

Various other metabolites arising from the synthesis and degradation ofestrogens are further modified by enzymatic sulfation or glucuronidationas a prerequisite for their clearance from the cell. Variation in thelevels of the enzymes that carry out these modifications may shift theintracellular concentrations of estrogen and its electrophilicmetabolites. For example, SULT1E1 is a member of the sulfotransferasefamily that preferentially sulfates estrone at the 3 position in adetoxification and clearance step. Another family of proteins, theUDP-glucuronosyltransferases (UGTs), participates in the clearance of awide variety of compounds, and includes UGT1A3 and UGT2b7, thesubstrates of which include estrone and 2-hydroxyestrone.

The forward and reverse conversion between catechol estrogens andcatechol estrogen quinones establishes the possibility of redox cycling,which results in continuous generation of superoxide anion (O₂ ⁻) Cellshave established strategies for detoxification of O₂ ⁻ produced byestrogen metabolism and other cellular processes. O₂ ⁻ is initiallyconverted to molecular oxygen (O₂)+hygrogen peroxide (H₂O₂), anotherROS, by a superoxide dismutase (SOD), which occur in cytoplamic (SOD1),mitochondrial (SOD2), and extracellular (SOD3) forms. The H202 producedby superoxide dismutase is further metabolized to H₂O₂ and molecularoxygen by catalase (CAT). The various enzymes of the peroxiredoxinfamily, including peroxiredoxins 2,3,4 and 6 (PRDX2, PRDX3, PRDX4 andPRDX6) also catalyze the inactivation of H₂O₂, as well as the reductionof organic hydroperoxides that may have been generated in the presenceof ROS. Glutathione peroxidases (GPX1 and GPX2) are also involved in thedetoxification of H₂O₂. Allelic variants of GPX1 have been associatedwith breast cancer risk (Knight et al., Cancer Epidemiol. BiomarkersPrev. 13: 146-9 (2004).

Hydrogen peroxide, in the presence of certain transition metal ions,gives rise to hydroxide ions, which not only can damage DNA directly butcan also initiate lipid peroxidation, giving rise to lipidhydroperoxides. These lipid hydroperoxides are believed to acceleratethe conversion of catechol estrogen to semiquinones and quinones bycytochrome P450 (Cavalieri CMLS), thus amplifying the production ofelectrophilic estrogen metabolites. Both peroxiredoxins (in addition toinactivating H202) and membrane-associated glutathione-S-transferases(in addition to conjugating glutathione to electrophilic estrogenmetabolites) can catalyze the reduction of organic hydroperoxides by theaction of ROS and therefore slow the procution of CE semiquinones and CEquinines.

The concentration of estrogen metabolites is affected by the rateestrogen synthesis as well as the routes and rates of degradation andclearance. Estrogen is synthesized from cholesterol via a complex seriesof reactions. Cholesterol is first metabolized in C21 steroid metabolismpathways to pregnenolone. As shown in FIG. 7, pregnenolone is thenconverted to androst-4-ene-3,17-dione by the action of a3β-hydroxysteroid dehydrogenase and a cytochrome P450 (CYP17A1) ineither order. Androst-4-ene-3,17-dione then gives rise to the keyestrogens, estrone and estradiol through the sequential actions of a17β-hydroxysteroid dehydrogenase (HSD17B1, HSD17B2, and HSD17B4) and thecytochrome P450 enzyme, aromatase (CYP19A1) in either order. Bothestrone and estradiol are subject to the degradation processes discussedabove.

Entry of estrogen precursors into the estrogen synthesis pathway can belimited by the alternate conversion of pregnenolone to progesterone andthen to 20α-hydroxyprogesterone by 20α-hydroxysteroid dehydrogenase(AKR1C3), reducing the amount of androst-4-ene-3,17-dione available forconversion to estrogens.

The Invention

The present invention takes the novel approach of measuring the mRNAexpression level of numerous genes that can affect the cellularconcentration of mutagenic estrogen metabolites at equilibrium, andidentifying markers of predisposition and prognosis in cancer thepathogenesis of which involves estrogen metabolism, such as breastcancer.

In particular, quantitative gene expression analysis performed inaccordance with the present invention resulted in the identification ofmolecular indicators of prognosis in cancer. Based on analysis of therelationship between gene expression in the sample set and DRFS, a setof genes has been identified, the expression levels of which areindicative of outcome after tumor resection and any accompanying therapywith tamoxifen and/or adjuvant chemotherapy. Outcome may be manifest invarious measurements including survival, recurrence-free survival anddistant recurrence-free survival (DRFS), all of which are within thescope of the invention.

The genes identified in accordance with the present invention, or anygene group formed by particular combination of such genes can be usedalone, or can be used together with one or more further diagnostic,prognostic and/or predictive indicators. Other diagnostic, prognosticand predictive indicators may include the expression of other genes orgene groups and may also include clinical variables including tumorsize, stage and grade. Other diagnostic, prognostic or predictiveindicators specifically include, individually or in any combination, thegenes and genes sets disclosed in any of the following PCT Publications:WO 2003/078,662; WO 2004/071,572; WO 2004/074,518; WO 2004/065,583; WO2004/111,273; WO 2004/111,603; WO 2005/008,213; WO 2005/040,396; WO2005/039,382; WO 2005/064,019.

Alone or in combination with other cancer markers, such as diagnostic,prognostic and/or predictive indicators, the genes and gene groups ofthe present invention can be used to calculate Recurrence Score, anaggregate indication, based on multiple prognostic indicators, of thelikelihood of a particular clinical outcome and/or drug responsiveness.Thus, for example, for an individual patient it is possible to provide aquantitative estimate of likelihood of outcome. This information can beutilized by the patient and treating physicians to make treatmentdecisions, in particular decisions regarding whether or not to treat thepatient with drugs that lead to appreciable adverse events.

In various embodiments of the inventions, various technologicalapproaches are available for determination of expression levels of thedisclosed genes, including, without limitation, RT-PCR, microarrays,serial analysis of gene expression (SAGE) and Gene Expression Analysisby Massively Parallel Signature Sequencing (MPSS), which will bediscussed in detail below. In particular embodiments, the expressionlevel of each gene may be determined in relation to various features ofthe expression products of the gene including exons, introns, proteinepitopes and protein activity. In other embodiments, the expressionlevel of a gene may be inferred from analysis of the structure of thegene, for example from the analysis of the methylation pattern of gene'spromoter(s).

B.2 Gene Expression Profiling

In general, methods of gene expression profiling can be divided into twolarge groups: methods based on hybridization analysis ofpolynucleotides, and methods based on sequencing of polynucleotides. Themost commonly used methods known in the art for the quantification ofmRNA expression in a sample include northern blotting and in situhybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283(1999)); RNAs e protection assays (Hod, Biotechniques 13:852-854(1992)); and reverse transcription polymerase chain reaction (RT-PCR)(Weis et al., Trends in Genetics 8:263-264 (1992)). Alternatively,antibodies may be employed that can recognize specific duplexes,including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes orDNA-protein duplexes. Representative methods for sequencing-based geneexpression analysis include Serial Analysis of Gene Expression (SAGE),and gene expression analysis by massively parallel signature sequencing(MPSS).

a. Reverse Transcriptase PCR (RT-PCR)

Of the techniques listed above, the most sensitive and most flexiblequantitative method is RT-PCR, which can be used to compare mRNA levelsin different sample populations, in normal and tumor tissues, with orwithout drug treatment, to characterize patterns of gene expression, todiscriminate between closely related mRNAs, and to analyze RNAstructure.

The first step is the isolation of mRNA from a target sample. Thestarting material is typically total RNA isolated from human tumors ortumor cell lines, and corresponding normal tissues or cell lines,respectively. Thus RNA can be isolated from a variety of primary tumors,including breast, lung, colon, prostate, brain, liver, kidney, pancreas,spleen, thymus, testis, ovary, uterus, etc., tumor, or tumor cell lines,with pooled DNA from healthy donors. If the source of mRNA is a primarytumor, mRNA can be extracted, for example, from frozen or archivedparaffin-embedded and fixed (e.g. formalin-fixed) tissue samples.

General methods for mRNA extraction are well known in the art and aredisclosed in standard textbooks of molecular biology, including Ausubelet al., Current Protocols of Molecular Biology, John Wiley and Sons(1997). Methods for RNA extraction from paraffin embedded tissues aredisclosed, for example, in Rupp and Locker, Lab Invest. 56:A (1987), andDe Andrés et al., BioTechniques 18:42044 (1995). In particular, RNAisolation can be performed using purification kit, buffer set andprotease from commercial manufacturers, such as Qiagen, according to themanufacturer's instructions. For example, total RNA from cells inculture can be isolated using Qiagen RNeasy mini-columns. Othercommercially available RNA isolation kits include MasterPure™ CompleteDNA and RNA Purification Kit (EPICENTRE®, Madison, Wis.), and ParaffinBlock RNA Isolation Kit (Ambion, Inc.). Total RNA from tissue samplescan be isolated using RNA Stat-60 (Tel-Test). RNA prepared from tumorcan be isolated, for example, by cesium chloride density gradientcentrifugation.

As RNA cannot serve as a template for PCR, the first step in geneexpression profiling by RT-PCR is the reverse transcription of the RNAtemplate into cDNA, followed by its exponential amplification in a PCRreaction. The two most commonly used reverse transcriptases are avilomyeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murineleukemia virus reverse transcriptase (MMLV-RT). The reversetranscription step is typically primed using specific primers, randomhexamers, or oligo-dT primers, depending on the circumstances and thegoal of expression profiling. For example, extracted RNA can bereverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, Calif.,USA), following the manufacturer's instructions. The derived cDNA canthen be used as a template in the subsequent PCR reaction.

Although the PCR step can use a variety of thermostable DNA-dependentDNA polymerases, it typically employs the Taq DNA polymerase, which hasa 5′-3′ nuclease activity but lacks a 3′-5′ proofreading endonucleaseactivity. Thus, TaqMan® PCR typically utilizes the 5′-nuclease activityof Taq or Tth polymerase to hydrolyze a hybridization probe bound to itstarget amplicon, but any enzyme with equivalent 5′ nuclease activity canbe used. Two oligonucleotide primers are used to generate an amplicontypical of a PCR reaction. A third oligonucleotide, or probe, isdesigned to detect nucleotide sequence located between the two PCRprimers. The probe is non-extendible by Taq DNA polymerase enzyme, andis labeled with a reporter fluorescent dye and a quencher fluorescentdye. Any laser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

TaqMan® RT-PCR can be performed using commercially available equipment,such as, for example, ABI PRISM 7700™ Sequence Detection System™(Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA), orLightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In apreferred embodiment, the 5′ nuclease procedure is run on a real-timequantitative PCR device such as the ABI PRISM 7700™ Sequence DetectionSystem™. The system consists of a thermocycler, laser, charge-coupleddevice (CCD), camera and computer. The system amplifies samples in a96-well format on a thermocycler. During amplification, laser-inducedfluorescent signal is detected at the CCD. The system includes softwarefor running the instrument and for analyzing the data.

5′-Nuclease assay data are initially expressed as C_(T), or thethreshold cycle. As discussed above, fluorescence values are recordedduring every cycle and represent the amount of product amplified to thatpoint in the amplification reaction. The point when the fluorescentsignal is first recorded as statistically significant is the thresholdcycle (C_(T)).

To minimize errors and the effect of sample-to-sample variation, RT-PCRis usually performed using one or more reference genes as internalstandards. The ideal internal standard is expressed at a constant levelamong different tissues, and is unaffected by the experimentaltreatment. RNAs most frequently used to normalize patterns of geneexpression are mRNAs for the housekeeping genesglyceraldehyde-3-phosphate-dehydrogenase (GAPD) and β-actin (ACTB).

A more recent variation of the RT-PCR technique is real timequantitative RT-PCR RT-PCR), which measures PCR product accumulationthrough a dual-labeled fluorigenic probe (i.e., TaqMan® probe). Realtime PCR is compatible both with quantitative competitive PCR, whereinternal competitor for each target sequence is used for normalization,and with quantitative comparative PCR using a normalization genecontained within the sample, or a housekeeping gene for RT-PCR. Forfurther details see, e.g. Held et al., Genome Research 6:986-994 (1996).

The steps of a representative protocol for profiling gene expressionusing fixed, paraffin-embedded tissues as the RNA source, including mRNAisolation, purification, primer extension and amplification are given invarious published journal articles (for example: T. E. Godfrey et al. J.Molec. Diagnostics 2: 84-91 (2000); K. Specht et al., Am. J. Pathol.158: 419-29 (2001); Cronin et al., Am J Pathol 164:35-42 (2004)}.Briefly, a representative process starts with cutting about 10 μm thicksections of paraffin-embedded tumor tissue samples. The RNA is thenextracted, and protein and DNA are removed. After analysis of the RNAconcentration, RNA repair and/or amplification steps may be included, ifnecessary, and RNA is reverse transcribed using gene specific promotersfollowed by RT-PCR.

b. Microarrays

Differential gene expression can also be identified, or confirmed usingthe microarray technique. Thus, the expression profile of breastcancer-associated genes can be measured in either fresh orparaffin-embedded tumor tissue, using microarray technology. In thismethod, polynucleotide sequences of interest (including cDNAs andoligonucleotides) are plated, or arrayed, on a microchip substrate. Thearrayed sequences are then hybridized with specific DNA probes fromcells or tissues of interest. Just as in the RT-PCR method, the sourceof mRNA typically is total RNA isolated from human tumors or tumor celllines, and corresponding normal tissues or cell lines. Thus RNA can beisolated from a variety of primary tumors or tumor cell lines. If thesource of mRNA is a primary tumor, mRNA can be extracted, for example,from frozen or archived paraffin-embedded and fixed (e.g.formalin-fixed) tissue samples, which are routinely prepared andpreserved in everyday clinical practice.

In a specific embodiment of the microarray technique, PCR amplifiedinserts of cDNA clones are applied to a substrate in a dense array.Preferably at least 10,000 nucleotide sequences are applied to thesubstrate. The microarrayed genes, immobilized on the microchip at10,000 elements each, are suitable for hybridization under stringentconditions. Fluorescently labeled cDNA probes may be generated throughincorporation of fluorescent nucleotides by reverse transcription of RNAextracted from tissues of interest. Labeled cDNA probes applied to thechip hybridize with specificity to each spot of DNA on the array. Afterstringent washing to remove non-specifically bound probes, the chip isscanned by confocal laser microscopy or by another detection method,such as a CCD camera. Quantitation of hybridization of each arrayedelement allows for assessment of corresponding mRNA abundance. With dualcolor fluorescence, separately labeled cDNA probes generated from twosources of RNA are hybridized pairwise to the array. The relativeabundance of the transcripts from the two sources corresponding to eachspecified gene is thus determined simultaneously. The miniaturized scaleof the hybridization affords a convenient and rapid evaluation of theexpression pattern for large numbers of genes. Such methods have beenshown to have the sensitivity required to detect rare transcripts, whichare expressed at a few copies per cell, and to reproducibly detect atleast approximately two-fold differences in the expression levels(Schena et al., Proc. Natl. Acad. Sci. USA 93(2):106-149 (1996)).Microarray analysis can be performed by commercially availableequipment, following manufacturer's protocols, such as by using theAffymetrix GenChip technology, or Incyte's microarray technology.

The development of microarray methods for large-scale analysis of geneexpression makes it possible to search systematically for molecularmarkers of cancer classification and outcome prediction in a variety oftumor types.

c. Serial Analysis of Gene Expression (SAGE)

Serial analysis of gene expression (SAGE) is a method that allows thesimultaneous and quantitative analysis of a large number of genetranscripts, without the need of providing an individual hybridizationprobe for, each transcript. First, a short sequence tag (about 10-14 bp)is generated that contains sufficient information to uniquely identify atranscript, provided that the tag is obtained from a unique positionwithin each transcript. Then, many transcripts are linked together toform long serial molecules, that can be sequenced, revealing theidentity of the multiple tags simultaneously. The expression pattern ofany population of transcripts can be quantitatively evaluated bydetermining the abundance of individual tags, and identifying the genecorresponding to each tag. For more details see, e.g. Velculescu et al.,Science 270:484-487 (1995); and Velculescu et al., Cell 88:243-51(1997).

d. Gene Expression Analysis by Massively Parallel Signature Sequencing(MPSS)

This method, described by Brenner et al., Nature Biotechnology18:630-634 (2000), is a sequencing approach that combines non-gel-basedsignature sequencing with in vitro cloning of millions of templates onseparate 5 μm diameter microbeads. First, a microbead library of DNAtemplates is constructed by in vitro cloning. This is followed by theassembly of a planar array of the template-containing microbeads in aflow cell at a high density (typically greater than 3×10⁶microbeads/cm²). The free ends of the cloned templates on each microbeadare analyzed simultaneously, using a fluorescence-based signaturesequencing method that does not require DNA fragment separation. Thismethod has been shown to simultaneously and accurately provide, in asingle operation, hundreds of thousands of gene signature sequences froma yeast cDNA library.

e. General Description of the mRNA Isolation, Purification andAmplification

The steps of a representative protocol for profiling gene expressionusing fixed, paraffin-embedded tissues as the RNA source, including mRNAisolation, purification, primer extension and amplification are providedin various published journal articles (for example: T. E. Godfrey etal,. J. Malec. Diagnostics 2: 84-91 [2000]; K. Specht et al., Am. J.Pathol. 158: 419-29 [2001]). Briefly, a representative process startswith cutting about 10 μm thick sections of paraffin-embedded tumortissue samples. The RNA is then extracted, and protein and DNA areremoved. After analysis of the RNA concentration, RNA repair and/oramplification steps may be included, if necessary, and RNA is reversetranscribed using gene specific-promoters followed by RT-PCR. Finally,the data are analyzed to identify the best treatment option(s) availableto the patient on the basis of the characteristic gene expressionpattern identified in the tumor sample examined, dependent on thepredicted likelihood of cancer recurrence.

f. Reference Gene Set

An important aspect of the present invention is to use the measuredexpression of certain genes by breast cancer tissue to provideprognostic or predictive information. For this purpose it is necessaryto correct for (normalize away) both differences in the amount of RNAassayed and variability in the quality of the RNA used. Well knownhousekeeping genes such as β-actin, GAPD, GUS, RPLO, and TFRC can beused as reference genes for normalization. Reference genes can also bechosen based on the relative invariability of their expression in thestudy samples and their lack of correlation with clinical outcome.Alternatively, normalization can be based on the mean or median signal(C_(T)) of all of the assayed genes or a large subset thereof (globalnormalization approach). Below, unless noted otherwise, gene expressionmeans normalized expression.

g. Primer and Probe Design

According to one aspect of the present invention, PCR primers and probesare designed based upon intron sequences present in the gene to beamplified. Accordingly, the first step in the primer/probe design is thedelineation of intron sequences within the genes. This can be done bypublicly available software, such as the DNA BLAT software developed byKent, W. J., Genome Res. 12(4):656-64 (2002), or by the BLAST softwareincluding its variations. Subsequent steps follow well establishedmethods of PCR primer and probe design.

In order to avoid non-specific signals, it is important to maskrepetitive sequences within the introns when designing the primers andprobes. This can be easily accomplished by using the Repeat Maskerprogram available on-line through the Baylor College of Medicine, whichscreens DNA sequences against a library of repetitive elements andreturns a query sequence in which the repetitive elements are masked.The masked intron sequences can then be used to design primer and probesequences using any commercially or otherwise publicly availableprimer/probe design packages, such as Primer Express (AppliedBiosystems); MGB assay-by-design (Applied Biosystems); Primer3 (SteveRozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general usersand for biologist programmers. In: Krawetz S, Misener S (eds)Bioinformatics Methods and Protocols: Methods in Molecular Biology.Humana Press, Totowa, N.J., pp 365-386).

The most important factors considered in PCR primer design includeprimer length, melting temperature (Tm), and G/C content, specificity,complementary primer sequences, and 3′-end sequence. In general, optimalPCR primers are generally 17-30 bases in length, and contain about20-80%, such as, for example, about 50-60% G+C bases. Tm's between 50and 80° C., e.g. about 50 to 70° C. are typically preferred.

For further guidelines for PCR primer and probe design see, e.g.Dieffenbach, C. W. et al., “General Concepts for PCR Primer Design” in:PCR Primer, A Laboratory Manual, Cold Spring Harbor Laboratory Press,New York, 1995, pp. 133-155; Innis and Gelfand, “Optimization of PCRs”in: PCR Protocols, A Guide to Methods and Applications, CRC Press,London, 1994, pp. 5-11; and Plasterer, T. N. Primerselect: Primer andprobe design. Methods Mol. Biol. 70:520-527 (1997), the entiredisclosures of which are hereby expressly incorporated by reference.

B.3 Sources of Biological Material

Treatment of cancer often involves resection of the tumor to the extentpossible without severely compromising the biological function of thepatient. As a result, tumor tissue is typically available for analysisfollowing initial treatment of the tumor, and this resected tumor hasmost often been the sample used in expression analysis studies.

Expression analysis can also be carried out on tumor tissue obtainedthrough other means such as core, fine needle, or other types of biopsy.

For particular tumor types, tumor tissue is appropriately obtained frombiological fluids using methods such as fine needle aspiration,bronchial lavage, or transbronchial biopsy.

Particularly in relatively advanced tumors, circulating tumor cells(CTC) are sometimes found in the blood of cancer patients. CTC recoveredfrom blood can also be used as a source of material for expressionanalysis.

Cellular constituents, including RNA and protein, derived from tumorcells have been found in biological fluids of cancer patients, includingblood and urine. Circulating nucleic acids and proteins may result fromtumor cell lysis and may be subjected to expression analysis.

B.3 Algorithms and Statistical Methods

When quantitative RT-PCR (qRT-PCR) is used to measure mRNA levels, mRNAamounts are expressed in C_(T) (threshold cycle) units (Held et al.,Genome Research 6:986-994 (1996)). The averaged sum of C_(T)s for thereference mRNAs is arbitrarily set (e.g. to zero), and each measuredtest mRNA C_(T) is given relative to this fixed reference. For example,if, for a particular patient tumor specimen the average of C_(T)s of thereference genes found to be 31 and C_(T) of test gene X is found to be35, the reported value for gene X is −4 (i.e. 31-35).

The normalized data can be used to analyze correlation between theexpression level of particular mRNAs and clinical outcome. Standardstatistical methods can be applied to identify those genes, for whichthe correlation between expression and outcome, in a univariateanalysis, is statistically significant. These genes are markers ofoutcome, given the existing clinical status. Multivariate analysis canbe applied to identify sets of genes, the expression levels of which,when used in combination, are better markers of outcome than theindividual genes that constitute the sets.

Further, it is possible to define groups of genes known or suspected tobe associated with particular aspects of the molecular pathology ofcancer. A gene can be assigned to a particular group based either on itsknown or suspected role in a particular aspect of the molecular biologyof cancer or based on its co-expression with another gene alreadyassigned to a particular group. Co-pending U.S. Patent Application60/561,035 defines several such groups and further shows that thedefinition of such groups (also termed axis or subset) is useful in thatit supports particular methods of data analysis and the elaboration ofmathematical algorithms, which in turn yields a more powerful predictorsof outcome than can be formulated if these groups are not defined.

In breast cancer, steroid metabolism, including synthesis anddegradation of steroids and clearance of intermediates is an aspect ofthe molecular pathology of cancer the importance of which has not beenadequately appreciated. Genes involved in steroid metabolism form a“Steroid Metabolism Group” the definition of which supports particularmethods of data analysis and will support the elaboration ofmathematical algorithms useful in the prediction of outcome in variousforms of cancer. The precise definition of the genes in the “SteroidMetabolism Group may vary depending on the identity of the steroidrelevant in a particular cancer but will be defined to include a) genes,the expression products of which are known or suspected to be involvedin synthesis and degradation of the particular steroid and clearance ofintermediates, and b) genes that are co-expressed with such genes.

B.5 Clinical Application of Data

The methods of this invention could be performed as a self-containedtest for cancer. Individual markers of the invention identified byunivariate analysis or sets of markers of the inventions (e.g.identified by multivariate analysis) are useful predictors of clinicaloutcome. Alternatively the markers can be applied as predictive elementsof a test that could include other predictive indicators including a)other genes and/or gene groups, or b) other clinical indicators such astumor stage and grade).

B.6 Kits of the Invention

The methods of this invention, when practiced for commercial diagnosticpurposes would typically be performed in a CLIA-approved clinicaldiagnostic laboratory. The materials for use in the methods of thepresent invention are suited for preparation of kits produced inaccordance with well known procedures. The invention thus provides kitsor components thereof, such kits comprising agents, which may includegene-specific or gene-selective probes and/or primers, for quantitatingthe expression of the disclosed genes for predicting prognostic outcomeor response to treatment. Such kits may optionally contain reagents forthe extraction of RNA from tumor samples, in particular fixedparaffin-embedded tissue samples and/or reagents for RNA amplification.In addition, the kits may optionally comprise the reagent(s) with anidentifying description or label or instructions relating to their usein the methods of the present invention. The kits may comprisecontainers (including microtiter plates suitable for use in an automatedimplementation of the method), each with one or more of the variousreagents (typically in concentrated form) utilized in the methods,including, for example, pre-fabricated microarrays, buffers, theappropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP and dTTP;or rATP, rCTP, rGTP and UTP), reverse transcriptase, DNA polymerase, RNApolymerase, and one or more probes and primers of the present invention(e.g., appropriate length poly(T) or random primers linked to a promoterreactive with the RNA polymerase). Mathematical algorithms used toestimate or quantify prognostic or predictive information are alsoproperly potential components of kits.

The methods provided by the present invention may also be automated inwhole or in part.

All aspects of the present invention may also be practiced such that alimited number of additional genes that are co-expressed with thedisclosed genes, for example as evidenced by high Pearson correlationcoefficients, are included in a prognostic or predictive test inaddition to and/or in place of disclosed genes.

Having described the invention, the same will be more readily understoodthrough reference to the following Example, which is provided by way ofillustration, and is not intended to limit the invention in any way.

EXAMPLES Example 1 Multiple GSTM1 Gene Family Members as Recurrance RiskMarkers

Breast Tumor FPE Specimens. Archival breast tumor FPE blocks, frompatients diagnosed between 1990 and 1997, were provided by ProvidenceSt. Joseph Medical Center, Burbank Calif. and were a subset of specimensexamined in a previously reported observational study [Esteban, J. etal. Prog. Proc Am Soc. Clin. Oncol. 22, 850 abstract (2003)]. The tumortissue specimens all came from female breast cancer patients withprimary disease (90% stage I or II) and relatively little nodalinvolvement (80% node negative). The protocol for use of these specimenswas approved by the IRB of that medical center.

Human genomic DNA samples. Genomic DNA was supplied by Dr. MaureenCronin. The samples were collected with informed consent for genotypingunder an IRB approved protocol.

RNA extraction and preparation. RNA was extracted from three 10 μm FPEsections per patient specimen according to Cronin et al. [Am. J. Pathol.164, 35-42 (2004)].

RNA amplification. The FPE RNA used in this study was amplified prior toRT-PCR assay in order to preserve the RNA for later studies. Fifty ng ofeach FPE RNA sample was amplified using the SenseAmp kit from Genisphere(Hatfield, Pa.). The amplified RNA products were purified using themirVana miRNA isolation kit from Ambion.

TaqMan primer/probe design: Exon-based assays: mRNA reference sequenceaccession numbers for genes of interest were identified and used toaccess the sequences through the NCBI Entrez Nucleotide database.Intron-based assays: Intron sequences were delineated by aligningappropriate mRNA reference sequences with their corresponding genes byusing the DNA BLAT software [Kent, W. J., Genome Res. 12,656-664(2002)].Repetitive sequences within the introns were identified and masked usingthe Repeat Masker program (Institute for Systems Biology). Primers andprobes were designed using Primer Express 2.0 (Applied Biosystems,Foster City, Calif.), or Primer 3 [Rozen, R. & Skaletsky, H. J. InKrawetz, S, Misener, S (eds) Bioinformatics Methods and Protocols:Methods in Molecular Biology: Humana Press, Totowa, N.J.,365-386(2000)]. Standard chemistry oligonucleotides were supplied byBiosearch Technologies Inc. (Novato, Calif.), Integrated DNATechnologies (Coralville, Iowa), and Eurogentech (San Diego, Calif.);MGB probes were supplied by Applied Biosystems. Amplicon sizes weretypically 60-85 bases in length. Fluorogenic probes were dual-labeledwith 5′-FAM and 3′-BHQ-2.

Reverse Transcription and TaqMan gene expression profiling RT-PCR wascarried out as previously described [Cronin et al., Am. J. Pathol. 164,35-42 (2004)].

Normalization and data analysis. Reference gene-based normalization wasused to correct for differences in RNA quality and total quantity of RNAassayed. A set of five reference genes were selected from a series ofcandidates based on their low variance in expression across all the FPEbreast cancer tissues and absence of a relationship (p>0.25) withdisease free survival. A reference CT for each tested tissue was definedas the average measured CT of the five reference genes. The normalizedmRNA level of a test gene within a tissue specimen was defined by thedifference between the average CT of the test gene (from triplicatemeasurements) minus the reference CT.

Statistical analysis. Least squares linear regression was used to modelthe relationship between the levels of pairs of assays. Pearson'scorrelation coefficient was used to summarize the strength of the linearrelationship. Cox Proportional Hazards regression was used to model therelationship between gene expression levels and disease-free survival,which was defined as the time from surgical removal of the breast tumoruntil the recurrence of breast cancer or death from breast cancer or anunknown cause.

The GSTM (GSMμ) gene family consists of five different closely relatedisotypes named GSTM1-GSTM5. We have reported four independent clinicalstudies in which GSTM gene expression strongly correlates with goodoutcome in primary breast cancer, based on measurements made using anRT-PCR probe-primer set (designated GSTM1.1) that was designed torecognize GSTM1 [8, Esteban, J. et al: Tumor gene expression andprognosis in breast cancer:multigene RT-PCR assay of paraffin-embeddedtissue. Prog. Proc Am Soc. Clin. Oncol. 22, 850 abstract (2003),Cobleigh, M. A. et al. Tumor gene expression predicts distantdisease-free survival (DDFS) in breast cancer patients with 10 or morepositive nodes: high throughput RT-PCR assay of paraffin-embedded tumortissues. Prog. Proc Am Soc. Clin. Oncol. 22, 850 abstract (2003), Paik,S. et al. Multi-gene RT-PCR assay for predicting recurrence in nodenegative breast cancer patients-NSABP studies B-20 and B-14. BreastCancer Res. Treat. 82:A16.abstract (2003)].

GSTM expression was examined by qRT-PCR in FPET primary breast cancertissues. GSTM1 was detected with the GSTM1.1 assay, which recognizesseveral GSTM isotypes. The estimate of relative risk in studies 1-4 wasbased on the hazard ratio (HR) from analysis of the time to breastcancer recurrence using univariate Cox proportional hazards regression.The estimate of relative risk in study 5 was based on the odds ratio(OR) from analysis of breast cancer death in a matched case-controlstudy using conditional logistic regression.

Study 1, Esteban et al., Prog. Proc Am Soc. Clin, Oncol. 22:850abstract, 2003; Study 2, Cobleigh et al., Clin Cancer Res11:8623-31,2005; Study 3, Paik et al., Breast Cancer Res. Treat. 82:A16abstract, 2003; Study 4, Paik et al, N Engl J Med 351:2817-26, 2004;Study 5, Habel et. al, Breast Cancer Res. Treat. 88:3019 abstract, 2004.The results are shown in Table 1.* Patients in studies 3-5 weretamoxifen treated, LN-,ER+. GSTM expression was a consistent predictorof favorable outcome in five independent breast cancer recurrencestudies.

TABLE 1 Relative Rank (among Total no. of Study Risk P-Value testedgenes) genes tested 1 Providence 0.71 0.0014 6 192 2 Rush 0.80 0.0200 5192 3 NSABP 20* 0.68 0.0005 7 192 4 NSABP 14* 0.73 <0.0001 5 21(OncotypeDX) 5 Kaiser* 0.72 <0.0010 ≦6 21 (OncotypeDX)

Sequence alignments of GSTM1 and GSTM2 amplicons with correspondingregions of other GSTM family members (FIG. 1). Sequences were aligned byClustal W (family member denoted in left column). Arrows mark forward(left) and reverse RT-PCR primer (right) regions. Sequences beneathhorizontal line indicates probe region. Gray boxes highlight mismatcheswith primers/probes in the first column. The vertical line in GSTM1.1indicates a spliced exon-exon junction. The vertical line in GSTM2int4.2indicates an unspliced intron-exon junction. In fact, alignment of thetargeted GSTM1 amplicon probe-primer set with homologous regions inGSTM2, GSTM4 and GSTM5 indicates only 1, 3 and 3 base mismatches,respectively, indicating that GSTM1.1 may also amplify those sequences(FIG. 1).

Consistent with the fact that 50% of the U.S. population is homozygousGSTM1-null, the GSTM1 intron-based assay displays a biphasic expressionpattern within 125 breast cancer specimens. FIG. 2 shows the number ofpatients (Y-axis) and corresponding Ct values (x-axis) were plotted forGSTM1.1, GSTM1int5.2 and GSTM2int4.2 assays. Expression levels weredetermined by TaqMan RT-PCR. “int” indicates that the assay was derivedfrom intron sequence.

It is noteworthy that a GSTM1.1 signal was detected in all specimens(C_(T)<40). This result is strong evidence that GSTM1.1 is not specificfor GSTM1, because it is well-established that approximately 50% of theCaucasian and Asian populations are homozygous null for the GSTM1 gene.FIG. 2 shows that in the case of GSTM1int5.2, RT-PCR signals distributein a bimodal pattern, with no signal detected in ˜50% of the specimens,consistent with specificity for GSTM1. GSTM1int3.1 showed a similarbimodal pattern as GSTM1int5. Furthermore, as shown in FIG. 3,genotyping of 22 independent human genomic DNA samples using GSTM1int5.2identified ˜50% as GSTM1 null, (C_(T)=40). C_(T) values were ˜31-32 forthe remaining samples. Again, GSTM1.1 failed to discriminate between thetwo GSTM1 genotypes, yielding C_(T)˜31-32 in all cases.

We also explored the expression of another GSTM isotype, GSTM2, using anintron-based design, designated GSTM2int4.2. This 73 base amplicondiffers from the other GSTM isotypes by 14 or more bases within thecorresponding primer/probe regions (FIG. 2). Expression of this sequencein the 125 patient specimens distributes across 6 C_(T) units, from34-40 (FIG. 2). Genotyping with GSTM2int4.2 gave uniform positivesignals for all 22 tested DNA specimens (FIG. 3) indicating that GSTM2is not deleted,

Pearson (R) correlation between GSTM family members. Table 2 shows thePearson (r) correlation for the various GStM gene family members asdetermined by various probe-primer sets. Bold font denotes R valuesbetween assay sets that we found to be specific for the designatedgenes. “int” indicates that the assay was derived from intron sequence.In general, the GSTM family members show positive correlations ofexpression. However, there is a wide range of correlations that vary notonly between genes but also between probe-primer sets within the samegene. Among the probe-primer sets thought to be gene specific (boldfont), correlations range from 0.15 to 0.91. GSTM1int3.1 and GSTM1int5.2showed the highest degree of co-expression (R=0.91). Interestingly,GSTM3.5 and GSTM3.6 show a more modest correlation (R=0.68) suggestingperhaps that they monitor alternate GSTM3 transcripts that aredifferentially regulated. GSTM4.1 vs. GSTM5.2 and GSTM4.1 vs.GSTM1int5.2show the lowest levels of coordinated expression (R=0.15-0.22) which wasnot unexpected since they are detecting transcripts from differentgenes. GSTM2int4.2 and GSTM3.6, the two genes that both contribute topositive prognosis in the multivariate analysis, show a modest positivecorrelation (0.42).

In summary, the positive effects of the GSTM family members are mostlikely due to a combination of protein function and co-expression.(Table 2).

TABLE 2 Pearson (R) GSTM1 int GSTM1 int GSTM2 int correlation 5.2 3.1GSTM1.1 4.2 GSTM3.6 GSTM4.1 GSTM5.2 GSTM1 int 1.00 5.2 GSTM1 int 0.911.00 3.1 GSTM1.1 0.52 0.49 1.00 GSTM2 int 0.26 0.25 0.57 1.00 4.2GSTM3.6 0.26 0.23 0.46 0.37 1.00 GSTM4.1 0.15 0.18 0.51 0.34 0.44 1.00GSTM5.2 N/A N/A 0.19 0.23 0.23 0.15 1.00 GSTM5.1 0.29 0.28 0.40 0.280.27 0.22 N/A

GSTM1-5 expression predict favorable outcome in the 125 breast cancerspecimen study. Multivariate analysis suggests that GSTM2 and GSTM3carry independent biomarker information. Univariate and multivariate CoxPH regression analysis. Assays are ordered by p-value, withp-values≦0.05 considered significant. Data in bold are assays that arespecific. “int” indicates that the assay was derived from intronsequence. (Tables 3 and 4).

The tables indicate that all 5 GSTM genes are indicators of positiveprognosis. The order of predictive strength from strongest to weakestis: GSTM3>GSTM2>GSTM4>GSTM5>GSTM1.

TABLE 3 Hazard HR HR Univariate Analysis Ratio 95% LCL 95% UCL P-ValueGSTM3.6 0.57 0.42 0.78 0.0003 GSTM2 int 4.2 0.64 0.49 0.83 0.0003GSTM1.1 0.71 0.58 0.86 0.0009 GSTM4.1 0.68 0.53 0.87 0.0044 GSTM1 int5.2 0.79 0.64 0.96 0.0128 GSTM5.2 0.77 0.58 1.02 0.0493 GSTM1 int 3.10.84 0.70 1.02 0.0632

A multivariate stepwise Cox PH analysis indicated that GSTM3 and GSTM2contributed independently to the positive prognosis (Table 4). Becausethere was an independent contribution to survival by both GSTM2 andGSTM3, it would suggest that each gene (product) has a biologicaleffect.

TABLE 4 Hazard HR HR Multivariate Analysis Ratio 95% LCL 95% UCL P-ValueGSTM3.6 0.65 0.47 0.90 0.0105 GSTM2 int 4.2 0.74 0.58 0.95 0.0185

The results indicate that all five GSTM genes are correlated with thelikelihood of breast cancer recurrence and suggest that certain GSTMfamily members contribute independent prognostic information.

Example 2 A Study of the Prognostic Value of GSTM Family Members andEstrogen Metabolizing Genes in Invasive Breast Cancer

The primary objective of this study was to determine the relationshipbetween the expression of genes involved in estrogen metabolism(including members of the GST gene family) and clinical outcome, inparticular distant recurrence-free survival (DRFS), in breast cancercarcinoma.

Study Design Inclusion Criteria

Samples were initially obtained from patients meeting the followingcriteria.

Surgery performed with diagnosis of invasive ductal carcinoma of thebreast, ductal carcinoma in situ (DCIS), lobular carcinoma of thebreast, or lobular carcinoma in situ (LCIS).

Histopathologic assessment indicating adequate amounts of tumor tissueand homogeneous pathology for inclusion in this research study.

For each patient sample included in the study, the expression level ofeach of 82 amplicons (shown in Table 5) was quantitatively assessedusing qRT-PCR and the correlation between gene expression and distantrecurrence-free survival (DRFS) for each of the test genes wasevaluated. Distant recurrence-free survival is the time from surgeryuntil the first diagnosis of distant recurrence. Contralateral disease,other second primary cancers, and deaths prior to distant recurrencewill be considered censoring events. For the primary analysis,ipsilateral breast recurrence, local chest wall recurrence and regionalrecurrence is ignored, i.e., not considered either as an event or acensoring event.

For this study one hundred twenty five (125) tumor samples were chosenfrom the patients. All recurring patients were included in the study, aswell as a randomly selected subset of patients who were censored (J.Esteban et al., ASCO Meeting Proceedings 22:850 (2003) (Abstract 3416)).

Gene Panel

A panel of genes potentially involved in metabolism or clearance ofestrogen or in other aspects of cancer pathophysiology was compiledbased on published literature. Analysis of 82 genes selected from thispanel or potentially useful as reference genes and listed in Table 5 wascarried out using quantitative RT-PCR. For certain of the genes,multiple probe primer ‘sets targeted to distinct gene sequences wereutilized. Gene names and primer and probe sequences used to quantifytranscript expression are listed in Table 6.

TABLE 5 Official NCBI Sequence Gene Symbol Sequence ID Version ID AKR1C1BC040210 BC040210.1 1645 AKR1C2 NM_001354 NM_001354.4 1646 AKR1C3NM_003739 NM_003739.4 8644 ATP5A1 NM_004046 NM_004046.4 498 ACTBNM_001101 NM_001101.2 60 BCL2 NM_000633 NM_000633.1 596 CAT NM_001752NM_001752.1 847 CD68 NM_001251 NM_001251.1 968 CDH1 NM_004360NM_004360.2 999 SCUBE2 NM_020974 NM_020974.1 57758 COMT NM_000754NM_000754.2 1312 COX8A NM_004074 NM_004074.2 1351 CRYZ NM_001889NM_001889.2 1429 CTSL2 NM_001333 NM_001333.2 1515 PPIH NM_006347NM_006347.3 10465 CYP17A1 NM_000102 NM_000102.2 1586 CYP19A1 NM_000103NM_000103.2 1588 CYP1A1 NM_000499 NM_000499.2 1543 CYP1B1 NM_000104NM_000104.2 1545 CYP4Z1 NM_178134 NM_178134.2 199974 EPHX1 NM_000120NM_000120.2 2052 ESR1 NM_000125 NM_000125.1 2099 FOXM1 NM_021953NM_021953.2 2305 GAPD NM_002046 NM_002046.2 2597 GCLC NM_001498NM_001498.2 2729 GCLM NM_002061 NM_002061.2 2730 GPX1 NM_000581NM_000581.2 2876 GPX2 NM_002083 NM_002083.2 2877 GSTM1 NM_000561NM_000561.2 2944 GSTM2 NM_000848 NM_000848.2 2946 GSTM3 NM_000849NM_000849.3 2947 GSTM4 NM_000850 NM_000850.3 2948 GSTM5 NM_000851NM_000851.2 2949 GSTP1 NM_000852 NM_000852.2 2950 GSTT1 NM_000853NM_000853.1 2952 GUSB NM_000181 NM_000181.1 2990 HOXB13 NM_006361NM_006361.2 10481 HSD17B1 NM_000413 NM_000413.1 3292 HSD17B2 NM_002153NM_002153.1 3294 HSD17B4 NM_000414 NM_000414.1 3295 IL17RB NM_018725NM_018725.2 55540 IMMT NM_006839 NM_006839.1 10989 MKI67 NM_002417NM_002417.2 4288 LIPA NM_000235 NM_000235.2 3988 MDH2 NM_005918NM_005918.2 4191 MGST1 NM_020300 NM_020300.3 4257 MGST3 NM_004528NM_004528.2 4259 MPV17 NM_002437 NM_002437.3 4358 MVP NM_017458NM_017458.2 9961 NAT1 NM_000662 NM_000662.4 9 NAT2 NM_000015 NM_000015.110 NCOA2 NM_006540 NM_006540.1 10499 NDUFA7 NM_005001 NM_005001.1 4701NQO1 NM_000903 NM_000903.1 1728 NQO2 NM_000904 NM_000904.1 4835 TP53NM_000546 NM_000546.2 7157 SERPINE1 NM_000602 NM_000602.1 5054 PGRNM_000926 NM_000926.2 5241 PRAME NM_006115 NM_006115.3 23532 PRDX2NM_005809 NM_005809.4 7001 PRDX3 NM_006793 NM_006793.2 10935 PRDX4NM_006406 NM_006406.1 10549 PRDX6 NM_004905 NM_004905.2 9588 RPLP0NM_001002 NM_001002.3 6175 SC5DL NM_006918 NM_006918.2 6309 SOD1NM_000454 NM_000454.4 6647 SOD2 NM_000636 NM_000636.1 6648 SOD3NM_003102 NM_003102.1 6649 SRD5A2 NM_000348 NM_000348.2 6716 STK6NM_003600 NM_003600.2 6790 SULT1E1 NM_005420 NM_005420.2 6783 SULT4A1NM_014351 NM_014351.2 25830 BIRC5 NM_001168 NM_001168.2 332 TBPNM_003194 NM_003194.2 6908 TFRC NM_003234 NM_003234.1 7037 TST NM_003312NM_003312.4 7263 UGT1A3 NM_019093 NM_019093.2 54659 UGT2B7 NM_001074NM_001074.1 7364 PLAU NM_002658 NM_002658.2 5328 VDAC1 NM_003374NM_003374.1 7416 VDAC2 NM_003375 NM_003375.2 7417 XPC NM_004628NM_004628.3 7508

TABLE 6 Oligo Probe Name Accession Number Reagent Oligo Sequence LengthAKR1C1.1 BC040210 Forward GTGTGTGAAGCTGAATGATGG 21 BC040210 ReverseCTCTGCAGGCGCATAGGT 18 BC040210 Probe CCAAATCCCAGGACAGGCATGAAG 24AKR1C2.1 NM_001354 Forward TGCCAGCTCATTGCTCTTAT 20 NM_001354 ReverseTCTGTCACTGGCCTGGTTAG 20 NM_001354 Probe CAAATGTTTCTTCCTCCCTCACAGGC 26AKR1C3.1 NM_003739 Forward GCTTTGCCTGATGTCTACCAGAA 23 NM_003739 ReverseGTCCAGTCACCGGCATAGAGA 21 NM_003739 Probe TGCGTCACCATCCACACACAGGG 23ATP5A1.1 NM_004046 Forward GATGCTGCCACTCAACAACT 20 NM_004046 ReverseTGTCCTTGCTTCAGCAACTC 20 NM_004046 Probe AGTTAGACGCACGCCACGACTCAA 24B-actin.2 NM_001101 Forward CAGCAGATGTGGATCAGCAAG 21 NM_001101 ReverseGCATTTGCGGTGGACGAT 18 NM_001101 Probe AGGAGTATGACGAGTCCGGCCCC 23 Bcl2.1NM_000633 Probe TGTACGGCCCCAGCATGCGG 20 NM_000633 ForwardCTGGGATGCCTTTGTGGAA 19 NM_000633 Reverse CAGAGACAGCCAGGAGAAATCA 22Bcl2.2 NM_000633 Forward CAGATGGACCTAGTACCCACTGAGA 25 NM_000633 ReverseCCTATGATTTAAGGGCATTTTTCC 24 NM_000633 Probe TTCCACGCCGAAGGACAGCGAT 22Bcl2 intron 1 NM_000633int1- Forward GCATCATTTGTTGGGTATGGAGTT 24 50 kb.150 kb NM_000633int1- Reverse TCTATGGAGGCCAATATTTGATTCT 25 50 kbNM_000633int1- Probe AGCCAGTGTCCCTCAACCCAACTTCTG 27 50 kb Bcl2 intron 1NM_000633int1- Forward GGGCAGTGGCCTGATGAA 18 50 kb.2 50 kbNM_000633int1- Reverse ATGGCAAAACTGTGTCTTTCCTTAT 25 50 kb NM_000633int1-Probe CTTTTCTTCATTTTTGCT 18 50 kb Bcl2 intron 1 NM_000633int1- ForwardGTCACTTTTATCTCACAGCATCACAA 26 100 kb.1 100 kb NM_000633int1- ReverseGCATTGGATCTTGGTGTCTTGA 22 100 kb NM_000633int1- ProbeAGGAACATCTGACAGCACTTGCCAGGTT 28 100 kb Bcl2 intron 1 NM_000633int1-Forward GGAGAAGTAGCCAGCCCATTTAA 23 150 kb.2 150 kb NM_000633int1-Reverse TGTCCCTGGCGCGTTTAG 18 150 kb NM_000633int1- ProbeATGTCAGCAAAGATTCCAGT 20 150 kb Bcl2 intron1 3′.1 NM_000633int1-3 ForwardCTAGCCACCCCCAAGAGAAAC 21 NM_000633int1-3 Reverse TGCCAACCTCTAAGGTCAAGGT22 NM_000633int1-3 Probe CCTGACAGCTCCCTTTCCCCAGGA 24 Bcl2-beta.1NM_000657 Forward TGGGTAGGTGCACTTGGTGAT 21 NM_000657 ReverseACTCCAACCCCCGCATCT 18 NM_000657 Probe ACCTGTGGCCTCAGCCCAGACTCA 24 CAT.1NM_001752 Forward ATCCATTCGATCTCACCAAGGT 22 NM_001752 ReverseTCCGGTTTAAGACCAGTTTACCA 23 NM_001752 Probe TGGCCTCACAAGGACTACCCTCTCATCC28 CD68.2 NM_001251 Forward TGGTTCCCAGCCCTGTGT 18 NM_001251 ReverseCTCCTCCACCCTGGGTTGT 19 NM_001251 Probe CTCCAAGCCCAGATTCAGATTCGAGTCA 28CDH1.3 NM_004360 Forward TGAGTGTCCCCCGGTATCTTC 21 NM_004360 ReverseCAGCCGCTTTCAGATTTTCAT 21 NM_004360 Probe TGCCAATCCCGATGAAATTGGAAATTT 27CEGP1.2 NM_020974 Forward TGACAATCAGCACACCTGCAT 21 NM_020974 ReverseTGTGACTACAGCCGTGATCCTTA 23 NM_020974 Probe CAGGCCCTCTTCCGAGCGGT 20CEGP1.6 NM_020974 Forward GCTGCATTTTATGTCCAAATGG 22 NM_020974 ReverseTGGTCTTGGGCATGGTTCA 19 NM_020974 Probe ATTTGTCCTTCCTCATTTTG 20CEGP1 intron 4.1 NM_020974 Forward TCCCCTTGCCTTTGGAGAA 19 NM_020974Reverse AAAGGCCTGGAGGCATCAA 19 NM_020974 Probe CAGCCCAAATCCT 13CEGP1 intron 5.1 NM_020974 Forward CTTAATGGTGTTTAGCAGAGATGCA 25NM_020974 Reverse CCACTGTAGCATGCGAAGCA 20 NM_020974 ProbeCAAATGCACAGGAAAC 16 COMT.1 NM_000754 Forward CCTTATCGGCTGGAACGAGTT 21NM_000754 Reverse CTCCTTGGTGTCACCCATGAG 21 NM_000754 ProbeCCTGCAGCCCATCCACAACCT 21 COX8.1 NM_004074 Forward CGTTCTGTCCCTCACACTGTGA22 NM_004074 Reverse CAAATGCAGTAACATGACCAGGAT 24 NM_004074 ProbeTGACCAGCCCCACCGGCC 18 CRYZ.1 NM_001889 Forward AAGTCCTGAAATTGCCATCA 20NM_001889 Reverse CACATGCATGGACCTTGATT 20 NM_001889 ProbeCCGATTCCAAAAGACCATCAGGTTCT 26 CTSL2.1 NM_001333 ForwardTGTCTCACTGAGCGAGCAGAA 21 NM_001333 Reverse ACCATTGCAGCCCTGATTG 19NM_001333 Probe CTTGAGGACGCGAACAGTCCACCA 24 CTSL2.10 NM_001333 ForwardTCAGAGGCTTGTTTGCTGAG 20 NM_001333 Reverse AGGACGAGCGAAAGATTCAT 20NM_001333 Probe CGACGGCTGCTGGTTTTGAAAC 22 CYP.1 NM_006347 ForwardTGGACTTCTAGTGATGAGAAAGATTGA 27 NM_006347 Reverse CACTGCGAGATCACCACAGGTA22 NM_006347 Probe TTCCCACAGGCCCCAACAATAAGCC 25 CYP17A1.1 NM_000102Forward CCGGAGTGACTCTATCACCA 20 NM_000102 Reverse GCCAGCATTGCCATTATCT 19NM_000102 Probe TGGACACACTGATGCAAGCCAAGA 24 CYP19A1.1 NM_000103 ForwardTCCTTATAGGTACTTTCAGCCATTTG 26 NM_000103 Reverse CACCATGGCGATGTACTTTCC 21NM_000103 Probe CACAGCCACGGGGCCCAAA 19 CYP1A1.2 NM_000499 ForwardAATAATTTCGGGGAGGTGGT 20 NM_000499 Reverse GGTTGGGTAGGTAGCGAAGA 20NM_000499 Probe TGGCTCTGGAAACCCAGCTGACTT 24 CYP1B1.3 NM_000104 ForwardCCAGCTTTGTGCCTGTCACTAT 22 NM_000104 Reverse GGGAATGTGGTAGCCCAAGA 20NM_000104 Probe CTCATGCCACCACTGCCAACACCTC 25 CYP4Z1.1 NM_178134 ForwardGCCTTACACCACGATGTGCAT 21 NM_176134 Reverse GTCGAGTAACCGGGATATGTTTACTAC27 NM_178134 Probe AAGGAATGCCTCCGCCTCTACGCAC 25 EPHX1.2 NM_000120Forward ACCGTAGGCTCTGCTCTGAA 20 NM_000120 Reverse TGGTCCAGGTGGAAAACTTC20 NM_000120 Probe AGGCAGCCAGACCCACAGGA 20 EstR1.1 NM_000125 ForwardCGTGGTGCCCCTCTATGAC 19 NM_000125 Reverse GGCTAGTGGGCGCATGTAG 19NM_000125 Probe CTGGAGATGCTGGACGCCC 19 FOXM1.1 NM_021953 ForwardCCACCCCGAGCAAATCTGT 19 NM_021953 Reverse AAATCCAGTCCCCCTACTTTGG 22NM_021953 Probe CCTGAATCCTGGAGGCTCACGCC 23 FOXM1.3 NM_021953 ForwardTGCCCAGATGTGCGCTATTA 20 NM_021953 Reverse TCAATGCCAGTCTCCCTGGTA 21NM_021953 Probe ATGTTTCTCTGATAATGTCC 20 FOXM1 intron 5.1 NM_021953Forward TGGACAGAGACAAGATGTGATGTG 24 NM_021953 ReverseGCTGGCACCTAGACAAAACATG 22 NM_021953 Probe CCATAGGGACCCTTC 15FOXM1 intron 7.1 NM_021953 Forward GGTGTCCTATTTTCCTCTGAAGAGA 25NM_021953 Reverse TGCAAGCTGAAGGTCCAACAT 21 NM_021953 ProbeTTCTGGCCAATTAAG 15 GAPDH.1 NM_002046 Forward ATTCCACCCATGGCAAATTC 20NM_002046 Reverse GATGGGATTTCCATTGATGACA 22 NM_002046 ProbeCCGTTCTCAGCCTTGACGGTGC 22 GCLC.3 NM_001498 Forward CTGTTGCAGGAAGGCATTGA20 NM_001498 Reverse GTCAGTGGGTCTCTAATAAAGAGATGAG 28 NM_901498 ProbeCATCTCCTGGCCCAGCATGTT 21 GCLM.2 NM_002061 ForwardTGTAGAATCAAACTCTTCATCATCAACTAG 30 NM_002061 ReverseCACAGAATCCAGCTGTGCAACT 22 NM_002061 Probe TGCAGTTGACATGGCCTGTTCAGTCC 26GPX1.2 NM_000581 Forward GCTTATGACCGACCCCAA 18 NM_000581 ReverseAAAGTTCCAGGCAACATCGT 20 NM_000581 Probe CTCATCACCTGGTCTCCGGTGTGT 24GPX2.2 NM_002083 Forward CACACAGATCTCCTACTCCATCCA 24 NM_002083 ReverseGGTCCAGCAGTGTCTCCTGAA 21 NM_002083 Probe CATGCTGCATCCTAAGGCTCCTCAGG 26GSTM1.1 NM_000561 Reverse GGCCCAGCTTGAATTTTTCA 20 NM_000561 ForwardAAGCTATGAGGAAAAGAAGTACACGAT 27 NM_000561 ProbeTCAGCCACTGGCTTCTGTCATAATCAGGAG 30 GSTM1 var2.1 NM_146421 ForwardCCATGGTTTGCAGGAAACAA 20 NM_146421 Reverse AGAACACAGGTCTTGGGAGGAA 22NM_146421 Probe ATCTCTGCCTACATGAAGTCCAGCC 25 GSTM1 intron 1.1 NM_000561Forward AACGGGTACGTGCAGTGTAAACT 23 NM_000561 Reverse GCAGGTCGCGTCAGAGATG19 NM_000561 Probe CCCTGACTTTGTCTGCACCAGGGAAG 26 GSTM1 intron 3.1NM_000561 Forward TCTGTGTCCACCTGCATTCG 20 NM_000561 ReverseCTGCTCATGGCAGGACTGAA 20 NM_000561 Probe TCATGTGACAGTATTCTTA 19GSTM1 intron 5.1 NM_000561int5 Forward CGACTCCAATGTCATGTCAACA 22NM_000561int5 Reverse ACCCTGGGATGCCTGGAT 18 NM_000561int5 ProbeAGAGGCAATTCCCACCAACCTTAGGACA 28 GSTM1 intron 5.2 NM_000561int5 ForwardGGCAATTCCCACCAACCTTA 20 NM_000561int5 ReverseAAACTTTACCATACAGGAACTGAATTTCT 29 NM_000561int5 ProbeACACGATCCAGGCATCCCAGGG 22 GSTM1 intron 5.3 NM_000561int5 ForwardATGGCACCCTCGAATTGC 18 NM_000561int5 Reverse TGCATGTCAATGACAGoACTCA 22NM_000561int5 Probe TCTTCTCCTCAACAGTTTT 19 GSTM1 intron 7.2NM_000561int7 Forward GCCTCCCTGTGGAAAAGGA 19 NM_000561int7 ReverseTCACACCAGGCCCTGTCA 18 NM_000561int7 Probe TCCTTGACTGCACAAACAG 19GSTM2 gene.1 NM_000848gene Forward GCAGGAACGAGAGGAGGAGAT 21NM_000848gene Reverse CAGCTCGGGTCAGAGATGGA 20 NM_000848gene ProbeCTCCCCTTGTGCAGAGTCGTCACAAA 26 GSTM2 gene.4 NM_000848gene ForwardCTGGGCTGTGAGGCTGAGA 19 NM_000848gene Reverse GCGAATCTGCTCCTTTTCTGA 21NM_000848gene Probe CCCGCCTACCCTCGTAAAGCAGATTCA 27 GSTM3.2 NM_000849Forward CAATGCCATCTTGCGCTACAT 21 NM_000849 ReverseGTCCACTCGAATCTTTTCTTCTTCA 25 NM_000849 Probe CTCGCAAGCACAACATGTGTGGTGAGA27 GSTM3.5 NM_000849 Forward CCAGAAGCCAAGGATCTCTCTAGT 24 NM_000849Reverse TATTCCTCCTGACATCACTGGGTAT 25 NM_000849 ProbeTGCCATTTGGGCCCTCTGACCAT 23 GSTM3.6 NM_000849 ForwardTCACAGTTTCCCTAGTCCTCGAA 23 NM_000849 Reverse CGAATATCCCAGTACCCGAGAA 22NM_000849 Probe CCCGTCACCATGTCGTGCGAGTC 23 GSTM4.1 NM_000850 ForwardCGGACCTTGCTCCCTGAAC 19 NM_000850 Reverse CGGAGCAGGTTGCTGGAT 18 NM_000850Probe AGTAAGATCCACCGCCACCTCCGAG 25 GSTM5.1 NM_000851 ForwardTCCCTGAGGCTCCCTTGACT 20 NM_000851 Reverse GGCTGTGGACAACAGAAGACAA 22NM_000851 Probe CCACCCACAATTCGAGCACAGTCCT 25 GSTM5.2 NM_000851 ForwardGAAAGGTGCTCTGTGCCAAGT 21 NM_000851 Reverse CCTAGCCCCTCTTTGAACCAT 21NM_000851 Probe ATTCGCGCTCCTGTAGGCCGTCTAGAA 27 GSTp.3 NM_000852 ForwardGAGACCCTGCTGTCCCAGAA 20 NM_000852 Reverse GGTTGTAGTCAGCGAAGGAGATC 23NM_000852 Probe TCCCACAATGAAGGTCTTGCCTCCCT 26 GSTT1.3 NM_000853 ForwardCACCATCCCCACCCTGTCT 19 NM_000853 Reverse GGCCTCAGTGTGCATCATTCT 21NM_000853 Probe CACAGCCGCCTGAAAGCCACAAT 23 GUS.1 NM_000181 ForwardCCCACTCAGTAGCCAAGTCA 20 NM_000181 Reverse CACGCAGGTGGTATCAGTCT 20NM_000181 Probe TCAAGTAAACGGGCTGTTTTCCAAACA 27 HOXB13.1 NM_006361Forward CGTGCCTTATGGTTACTTTGG 21 NM_006361 Reverse CACAGGGTTTCAGCGAGC 18NM_006361 Probe ACACTCGGCAGGAGTAGTACCCGC 24 HSD17B1.1 NM_000413 ForwardCTGGACCGCACGGACATC 18 NM_000413 Reverse CGCCTCGCGAAAGACTTG 18 NM_000413Probe ACCGCTTCTACCAATACCTCGCCCA 25 HSD17B2.1 NM_002153 ForwardGCTTTCCAAGTGGGGAATTA 20 NM_002153 Reverse TGCCTGCGATATTTGTTAGG 20NM_002153 Probe AGTTGCTTCCATCCAACCTGGAGG 24 HSD17B4.1 NM_000414 ForwardTTGTCCTTTGGCTTTGTCAC 20 NM_000414 Reverse CAATCCATCCTGCTCCAAC 19NM_000414 Probe CAAACAAGCCACCATTCTCCTCACA 25 IL17RB.2 NM_018725 ForwardACCCTCTGGTGGTAAATGGA 20 NM_018725 Reverse GGCCCCAATGAAATAGACTG 20NM_018725 Probe TCGGCTTCCCTGTAGAGCTGAACA 24 IMMT.1 NM_006839 ForwardCTGCCTATGCCAGACTCAGA 20 NM_006839 Reverse GCTTTTCTGGCTTCCTCTTC 20NM_006839 Probe CAACTGCATGGCTCTGAACAGCCT 24 Ki-67.2 NM_002417 ForwardCGGACTTTGGGTGCGACTT 19 NM_002417 Reverse TTACAACTCTTCCACTGGGACGAT 24NM_002417 Probe CCACTTGTCGAACCACCGCTCGT 23 LIPA.1 NM_000235 ForwardCCAGTTGTCTTCCTGCAACA 20 NM_000235 Reverse CTGTTGGCAAGGTTTGTGAC 20NM_000235 Probe CCAGTTACTAGAATCTGCCAGCAAGCCA 28 MDH2.1 NM_005918 ForwardCCAACACCTTTGTTGCAGAG 20 NM_005918 Reverse CAATGACAGGGACGTTGACT 20NM_005918 Probe CGAGCTGGATCCAAACCCTTCAG 23 MGST1.2 NM_020300 ForwardACGGATCTACCACACCATTGC 21 NM_020300 Reverse TCCATATCCAACAAAAAAACTCAAAG 26NM_020300 Probe TTTGACACCCCTTCCCCAGCCA 22 MGST3.1 NM_004528 ForwardAGCTGTTGGAGGTGTTTACCA 21 NM_004528 Reverse TCGTCCAACAATCCAGGC 18NM_004528 Probe AAGCCCAGGCCAGAAGCTATACGC 24 MMTV-like env.3 AF346816Forward CCATACGTGCTGCTACCTGT 20 AF346816 Reverse CCTAAAGGTTTGAATGGCAGA21 AF346816 Probe TCATCAAACCATGGTTCATCACCAATATC 29 MPV17.1 NM_002437Forward CCAATGTGTTGCTGTTATCTGGAA 24 NM_002437 ReverseATGGAGTGAGGCAGGCTTAGAG 22 NM_002437 Probe TCCTACCTGTCCTGGAAGGCACATCG 26MVP.1 NM_017458 Forward ACGAGAACGAGGGCATCTATGT 22 NM_017458 ReverseGCATGTAGGTGCTTCCAATCAC 22 NM_017458 Probe CGCACCTTTCCGGTCTTGACATCCT 25NAT1.1 NM_000662 Forward TGGTTTTGAGACCACGATGT 20 NM_000662 ReverseTGAATCATGCCAGTGCTGTA 20 NM_000662 Probe TGGAGTGCTGTAAACATACCCTCCCA 26NAT2.1 NM_000015 Forward TAACTGACATTCTTGAGCACCAGAT 25 NM_000015 ReverseATGGCTTGCCCACAATGC 18 NM_000015 Probe CGGGCTGTTCCCTTTGAGAACCTTAACA 28NCOA2.1 NM_006540 Forward AGTGACCTCCGTGCCTACGT 20 NM_006540 ReverseCTCCCCTCAGAGCAGGATCA 20 NM_006540 Probe CCTCCATGGGTCCCGAGCAGG 21NDUFA7.1 NM_005001 Forward GCAGCTACGCTACCAGGAG 19 NM_005001 ReverseGGAGAGCTTGTGGCTAGGAC 20 NM_005001 Probe TCTCCAAGCGAACTCAGCCTCCTC 24NQO1.1 NM_000903 Forward CAGCAGACGCCCGAATTC 18 NM_000903 ReverseTGGTGTCTCATCCCAAATATTCTC 24 NM_000903 Probe AGGCGTTTCTTCCATCCTTCCAGGATT27 NQO2.1 NM_000904 Forward AGCGCTCCTTTCCGTAACC 19 NM_000904 ReverseTCCATTGACTCCTUCTTCGTGTA 24 NM_000904 Probe ATCTCGGCCGTGCCTCCCG 19 P53.2NM_000546 Forward CTTTGAACCCTTGCTTGCAA 20 NM_000546 ReverseCCCGGGACAAAGCAAATG 18 NM_000546 Probe AAGTCCTGGGTGCTTCTGACGCACA 25PAI1.3 NM_000602 Forward CCGCAACGTGGTTTTCTCA 19 NM_000602 ReverseTGCTGGGTTTCTCCTCCTGTT 21 NM_000602 Probe CTCGGTGTTGGCCATGCTCCAG 22 PR.6NM_000926 Forward GCATCAGGCTGTCATTATGG 20 NM_000926 ReverseAGTAGTTGTGCTGCCCTTCC 20 NM_000926 Probe TGTCCTTACCTGTGGGAGCTGTAAGGTC 28PR.12 NM_000926 Forward GTTCCATCCCAAAGAACCTG 20 NM_000926 ReverseGAAACTCTGGAGTTGGCATTT 21 NM_000926 Probe CCACCCGTTATTCTGAATGCTACTCTCA 28PRAME.3 NM_006115 Forward TCTCCATATCTGCCTTGCAGAGT 23 NM_006115 ReverseGCACGTGGGTCAGATTGCT 19 NM_006115 Probe TCCTGCAGCACCTCATCGGGCT 22 PRAME.4NM_006115 Forward CCACTGCTCCCAGCTTACAAC 21 NM_006115 ReverseCTGCAAGGCAGATATGGAGATG 22 NM_006115 Probe AATTCCCGTAGAAGCTTAA 19PRAME intron 5.1 NM_006115 Forward ATCAGGCACAGAGATAGAGGTGACT 25NM_006115 Reverse TCTTTCAACTCGGGCTTCCTT 21 NM_006115 ProbeCCCAGGCAGTGGCA 14 PRDX2.1 NM_005809 Forward GGTGTCCTTCGCCAGATCAC 20NM_005809 Reverse CAGCCGCAGAGCCTCATC 18 NM_005809 ProbeTTAATGATTTGCCTGTGGGACGCTCC 26 PRDX3.1 NM_006793 ForwardTGACCCCAATGGAGTCATCA 20 NM_006793 Reverse CCAAGCGGAGGGTTTCTTC 19NM_006793 Probe CATTTGAGCGTCAACGATCTCCCAGTG 27 PRDX4.1 NM_006406 ForwardTTACCCATTTGGCCTGGATTAA 22 NM_006406 Reverse CTGAAAGAAGTGGAATCCTTATTGG 25NM_006406 Probe CCAAGTCCTCCTTGTCTTCGAGGGGT 26 PRDX6.1 NM_004905 ForwardCTGTGAGCCAGAGGATGTCA 20 NM_004905 Reverse TGTGATGACACCAGGATGTG 20NM_004905 Probe CTGCCAATTGTGTTTTCCTGCAGC 24 RPLPO.2 NM_001002 ForwardCCATTCTATCATCAACGGGTACAA 24 NM_001002 Reverse TCAGCAAGTGGGAAGGTGTAATC 23NM_001002 Probe TCTCCACAGACAAGGCCAGGACTCG 25 SC5DL.1 NM_006918 ForwardCGCCTACATAAACCTCACCA 20 NM_006918 Reverse CCATCAATAGGGTGAAAAGCA 21NM_006918 Probe TGGAAGATTCCTACTCCATTTGCAAGTCA 29 SOD1.1 NM_000454Forward TGAAGAGAGGCATGTTGGAG 20 NM_000454 Reverse AATAGACACATCGGCCACAC20 NM_000454 Probe TTTGTCAGCAGTCACATTGCCCAA 24 SOD2.1 NM_000636 ForwardGCTTGTCCAAATCAGGATCCA 21 NM_000636 Reverse AGCGTGCTCCCACACATCA 19NM_000636 Probe AACAACAGGCCTTATTCCACTGCTGGG 27 SOD3.1 NM_003102 ForwardCCATAAGCCCTGAGACTCCC 20 NM_003102 Reverse TAGGAGGAACCTGAAGGCG 19NM_003102 Probe TTGACCTGACGATCTTCCCCCTTC 24 SRD5A2.1 NM_000348 ForwardGTAGGTCTCCTGGCGTTCTG 20 NM_000348 Reverse TCCCTGGAAGGGTAGGAGTAA 21NM_000348 Probe AGACACCACTCAGAATCCCCAGGC 24 STK15.2 NM_003600 ForwardCATCTTCCAGGAGGACCACT 20 NM_003600 Reverse TCCGACCTTCAATCATTTCA 20NM_003600 Probe CTCTGTGGCACCCTGGACTACCTG 24 STK15.8 NM_003600 ForwardGCCCCCTGAAATGATTGAAG 20 NM_003600 Reverse TCCAAGGCTCCAGAGATCCA 20NM_003600 Probe TTCTCATCATGCATCCGA 18 STK15 intron 2.1 NM_003600 ForwardCATTCACATTTATAAACCCACATGGA 26 NM_003600 Reverse AATCCAAAGTAAAGGCGGAAAGA23 NM_003600 Probe TGGTCTTGTCGGGAAT 16 STK15 intron 4.1 NM_003600Forward GCGAGGAATGAACCCACAGA 20 NM_003600 ReverseGCATGAGAACCAGTGGATTTAGACT 25 NM_003600 Probe CGCTAAAAGCAAAAGA 16SULT1E1.1 NM_005420 Forward ATGGTGGCTGGTCATCCAA 19 NM_005420 ReverseATAAGGAACCTGTCCTTGCATGAA 24 NM_005420 ProbeTTCTCCACAAACTCTGGAAAGGATCCAGGA 30 SULT4A1.1 NM_014351 ForwardCACCTGCCCTACCGCTTTC 19 NM_014351 Reverse GGGTTGCGAGCCATATAGATG 21NM_014351 Probe CCTCTGACCTCCACAATGGAGACTCCA 27 SURV.2 NM_001168 ForwardTGTTTTGATTCCCGGGCTTA 20 NM_001168 Reverse CAAAGCTGTCAGCTCTAGCAAAAG 24NM_001168 Probe TGCCTTCTTCCTCCCTCACTTCTCACCT 28 TBP.1 NM_003194 ForwardGCCCGAAACGCCGAATATA 19 NM_003194 Reverse CGTGGCTCTCTTATCCTCATGAT 23NM_003194 Probe TACCGCAGCAAACCGCTTGGG 21 TFRC.3 NM_003234 ForwardGCCAACTGCTTTCATTTGTG 20 NM_003234 Reverse ACTCAGGCCCATTTCCTTTA 20NM_003234 Probe AGGGATCTGAACCAATACAGAGCAGACA 28 TST.1 NM_003312 ForwardGGAGCCGGATGCAGTAGGA 19 NM_003312 Reverse AAGTCCATGAAAGGCATGTTGA 22NM_003312 Probe ACCACGGATATGGCCCGAGTCCA 23 UGT1A3.1 NM_019093 ForwardGATGCCCTTGTTTGGTGATCA 21 NM_019093 Reverse AGGGTCACTCCAGCTCCCTTA 21NM_019093 Probe TCTCCATGCGCTTTGCATTGTCCA 24 UGT2B7.2 NM_001074 ForwardCAATGGCATCTACGAGGCA 19 NM_001074 Reverse CAGGTTGATCGGCAAACA 18 NM_001074Probe AATCCCCACCATAGGGATCCCATG 24 upa.3 NM_002658 ForwardGTGGATGTGCCCTGAAGGA 19 NM_002658 Reverse CTGCGGATCCAGGGTAAGAA 20NM_002658 Probe AAGCCAGGCGTCTACACGAGAGTCTCAC 28 VDAC1.1 NM_003374Forward GCTGCGACATGGATTTCGA 19 NM_003374 Reverse CCAGCCCTCGTAACCTAGCA 20NM_003374 Probe TTGCTGGGCCTTCCATCCGG 20 VDAC2.1 NM_003375 ForwardACCCACGGACAGACTTGC 18 NM_003375 Reverse AGCTTTGCCAAGGTCAGC 18 NM_003375Probe CGCGTCCAATGTGTATTCCTCCAT 24 XPC.1 NM_004628 ForwardGATACATCGTCTGCGAGGAA 20 NM_004628 Reverse CTTTCAATGACTGCCTGCTC 20NM_004628 Probe TTCAAAGACGTGCTCCTGACTGCC 24

TABLE 7 Amplicon  Accession  Amplicon Sequence Name Number AKR1C1.1BC040210AGATGAGAGCAGCCTGAACTTACACTGTGAAAATGCCCTGGAGAAATGCAGAGATGCAGGTTTAATGAAGTCCATCAAKR1C2.1 NM_001354TGCCAGCTCATTGCTCTTATAGCCTGTGAGGGAGGAAGAAACATTTGCTAACCAGGCCAGTGACAGAAKR1C3.1 NM_003739GCTTTGCCTGATGTCTACCAGAAGCCCTGTGTGTGGATGGTGACGCAGAGGACGTCTCTATGCCGGTGACTGGACATP5A1.1 NM_004046GATGCTGCCACTCAACAACTTTTGAGTCGTGGCGTGCGTCTAACTGAGTTGCTGAAGCAAGGACAB-actin.2 NM_001101CAGCAGATGTGGATCAGCAAGCAGGAGTATGACGAGTCCGGCCCCTCCATCGTCCACCGCAAATGCBcl2.1 NM_000633CTGGGATGCCTTTGTGGAACTGTACGGCCCCAGCATGCGGCCTCTGTTTGATTTCTCCTGGCTGTCTCTGBcl2.2 NM_000633CAGATGGACCTAGTACCCACTGAGATTTCCACGCCGAAGGACAGCGATGGGAAAAATGCCCTTAAATCATAGGBcl2  NM_000633int1-GCATCATTTGTTGGGTATGGAGTTGCAGAAGTTGGGTTGAGGGACACTGGCTTCTAGAATCAAATATTGGCCTCCATintron 1 50 kb AGA 50 kb.1 Bcl2  NM_000633int1-GGGCAGTGGCCTGATGAAAAGCAAAAATGAAGAAAAGAATAAGGAAAGACACAGTTTTGCCAT intron 150 kb 50 kb.2 Bcl2  NM_000633int1-GTCACTTTTATCTCACAGCATCACAAGGAGGAACATCTGACAGCACTTGCCAGGTTATCAAGACACCAAGATCCAATintron 1 100 kb GC 100 kb.1 Bcl2  NM_000633int1-GGAGAAGTAGCCAGCCCATTTAAAATGTCAGCAAAGATTCCAGTTGTCTAAACGCGCCAGGGACAintron 1 150 kb 150 kb.2 Bcl2  NM_000633int1-CTAGCCACCCCCAAGAGAAACCCCCTGACAGCTCCCTTTCCCCAGGAGAACCTTGACCTTAGAGGTTGGCAintron1 3 3′.1 Bcl2- NM_000657TGGGTAGGTGCACTTGGTGATGTGAGTCTGGGCTGAGGCCACAGGTCCGAGATGCGGGGGTTGGAGTbeta.1 CAT.1 NM_001752ATCCATTCGATCTCACCAAGGTTTGGCCTCACAAGGACTACCCTCTCATCCCAGTTGGTAAACTGGTCTTAAACCGGA CD68.2 NM_001251TGGTTCCCAGCCCTGTGTCCACCTCCAAGCCCAGATTCAGATTCGAGTCATGTACACAACCCAGGGTGGAGGAGCDH1.3 NM_004360TGAGTGTCCCCCGGTATCTTCCCCGCCCTGCCAATCCCGATGAAATTGGAAATTTTATTGATGAAAATCTGAAAGCGGCTG CEGP1.2 NM_020974TGACAATCAGCACACCTGCATTCACCGCTCGGAAGAGGGCCTGAGCTGCATGAATAAGGATCACGGCTGTAGTCACACEGP1.6 NM_020974GCTGCATTTTATGTCCAAATGGAACCTTCCAAAATGAGGAAGGACAAATGACTTGTGAACCATGCCCAAGACCACEGP1  NM_020974int4 TCCCCTTGCCTTTGGAGAACAGCCCAAATCCTTTGATGCCTCCAGGCCTTTintron 4.1 CEGP1  NM_020974int5CTTAATGGTGTTTAGCACAGATGCAGGCTGTTTCCTGTGCATTTGCCCCCCCAGCAGGCCCTGTGCTGCTTCGCATGintron 5.1 CTACAGTGG COMT.1 NM_000754CCTTATCGGCTGGAACGAGTTCATCCTGCAGCCCATCCACAACCTGCTCATGGGTGACACCAAGGAGCOX8.1 NM_004074CGTTCTGTCCCTCACACTGTGACCTGACCAGCCCCACCGGCCCATCCTGGTCATGTTACTGCATTTGCRYZ.1 NM_001889AAGTCCTGAAATTGCGATCAGATATTGCAGTACCGATTCCAAAAGACCATCAGGTTCTAATCAAGGTCCATGCATGTG CTSL.2.1 NM_001333TGTCTCACTGAGCGAGCAGAATCTGGTGGACTGTTCGCGTCCTCAAGGCAATCAGGGCTGCAATGGTCTSL2.10 NM_001333TCAGAGGCTTGTTTGCTGAGGGTGCCTGCGCAGCTGCGACGGCTGCTGGTTTTGAAACATGAATCTTTCGCTCGTCCT CYP.1 NM_006347TGGACTTCTAGTGATGAGAAAGATTGAGAATGTTCCCACAGGCCCCAACAATAAGCCCAAGCTACCTGTGGTGATCTCGCAGTG CYP17A1.1 NM_000102CCGGAGTGACTCTATCACCAACATGCTGGACACACTGATGCAAGCCAAGATGAACTCAGATAATGGCAATGCTGGCCYP19A1.1 NM_000103TCCTTATAGGTACTTTCAGCCATTTGGCTTTGGGCCCCGTGGCTGTGCAGGAAAGTACATCGCCATGGTGCYP1A1.2 NM_000499AATAATTTCGGGGAGGTGGTTGGCTCTGGAAACCCAGCTGACTTCATCCCTATTCTTCGCTACCTACCCAACCCYP1B1.3 NM_000104CCAGCTTTGTGCCTGTCACTATTCCTCATGCCACCACTGCCAACACCTCTGTCTTGGGCTACCACATTCCCCYP4Z1.1 NM_178134GCCTTACACCACGATGTGCATCAAGGAATGCCTCCGCCTCTACGCACCGGTAGTAAACATATCCCGGTTACTCGACEPHX1.2 NM_000120ACCGTAGGCTCTGCTCTGAATGACTCTCCTGTGGGTCTGGCTGCCTATATTCTAGAGAAGTTTTCCACCTGGACCAEstR1.1 NM_000125CGTGGTGCCCCTCTATGACCTGCTGCTGGAGATGCTGGACGCCCACCGCCTACATGCGCCCACTAGCCFOXM1.1 NM_021953CCACCCCGAGCAAATCTGTCCTCCCCAGAACCCCTGAATCCTGGAGGCTCACGCCCCCAGCCAAAGTAGGGGGACTGGATTT FOXM1.3 NM_021953TGCCCAGATGTGCGCTATTAGATGTTTCTCTGATAATGTCCCCAATCATACCAGGGAGACTGGCATTGAFOXM1  NM_021953TGGACAGAGACAAGATGTGATGTGGGGAAGGGTCCCTATGGCCATGTTTTGTCTAGGTGCCAGCintron 5.1 int5 FOXM1  NM_021953GGTGTCCTATTTTCCTCTGAAGAGAGATTCTGGCCAATTAAGAATGTTGGACCTTCAGCTTGCAintron 7.1 int7 GAPDH.1 NM_002046ATTCCACCCATGGCAAATTCCATGGCACCGTCAAGGCTGAGAACGGGAAGCTTGTCATCAATGGAAATCCCATCGCLC.3 NM_001498CTGTTGCAGGAAGGCATTGATCATCTCCTGGCCCAGCATGTTGCTCATCTCTTTATTAGAGACCCACTGACGCLM.2 NM_002061TGTAGAATCAAACTCTTCATCATCAACTAGAAGTGCAGTTGACATGGCCTGTTCAGTCCTTGGAGTTGCACAGCTGGATTCTGTG GPX1.2 NM_000581GCTTATGACCGACCCCAAGCTCATCACCTGGTCTCCGGTGTGTCGCAACGATGTTGCCTGGAACTTTGPX2.2 NM_002083CACACAGATCTCCTACTCCATCCAGTCCTGAGGAGCCTTAGGATGCAGCATGCCTTCAGGAGACACTGCTGGACCGSTM1.1 NM_000561AAGCTATGAGGAAAAGAAGTACACGATGGGGGACGCTCCTGATTATGACAGAAGCCAGTGGCTGAATGAAAAATTCAAGCTGGGCC GSTM1  NM_146421CCATGGTTTGCAGGAAACAAGGGCTTGGAGAAGATCTCTGCCTACATGAAGTCCAGCCGCTTCCTCCCAAGACCTGTvar2.1 GTTCT GSTM1  NM_000561int1AACGGGTACGTGCAGTGTAAACTGGGGGCTTCCCTGGTGCAGACAAAGTCAGGGACCCTCCATCTCTGACGCGACCTintron 1.1 GC GSTM1  NM_000561int3TCTGTGTCCACCTGCATTCGTTCATGTGACAGTATTCTTATTTCAGTCCTGCCATGAGCAG intron 3.1GSTM1  NM_000561int5CGACTCCAATGTCATGTCAACAAAAGCAGAGGCAATTCCCACCAACCTTAGGACACGATCCAGGCATCCCAGGGTintron 5.1 GSTM1  NM_000561int5GGCAATTCCCACCAACCTTAGGACACGATCCAGGCATCCCAGGGTAGAAATTCAGTTCCTGTATGGTAAAGTTTintron 5.2 GSTM1  NM_000561int5ATGGCACCCTCGAATTGCATCTTCTCCTCAACAGTTTTCTGAGTGCTGTCATTGACATGCA intron 5.3GSTM1  NM_000561int7GCCTCCCTGTGGAAAAGGAGACTGTTTGTGCAGTCAAGGAGTGACAGGGCCTGGTGTGA intron 7.2GSTM2  NM_000848geneGCAGGAACGAGAGGAGGAGATGGGGCTCCCCTTGTGCAGAGTCGTCACAAAGTCAGGGACCCTCCATCTCTGACCCGgene.1 AGCTG GSTM2  NM_000848geneCTGGGCTGTGAGGCTGAGAGTGAATCTGCTTTACGAGGGTAGGCGGGGAATCAGAAAAGGAGCAGATTCGCgene.4 GSTM3.2 NM_000849CAATGCCATCTTGCGCTACATCGCTCGCAAGCACAACATGTGTGGTGAGACTGAAGAAGAAAAGATTCGAGTGGACGSTM3.5 NM_000849CCAGAAGCCAAGGATCTCTCTAGTGATGGTCAGAGGGCCCAAATGGCAGGGATACCCAGTGATGTCAGGAGGAATAGSTM3.6 NM_003849TCACAGTTTCCCTAGTCCTCGAAGGCTCGGAAGCCCGTCACCATGTCGTGCGAGTCGTCTATGGTTCTCGGGTACTGGGATATTCG GSTM4.1 NM_000850CGGACCTTGCTCCCTGAACACTCGGAGGTGGCGGTGGATCTTACTCCTTCCAGCCAGTGAGGATCCAGCAACCTGCTCCG GSTM5.1 NM_000851TCCCTGAGGCTCCCTTGACTCAGGACTGTGCTCGAATTGTGGGTGGTTTTTTGTCTTCTGTTGTCCACAGCCGSTM5.2 NM_000851GAAAGGTGCTCTGTGCCAAGTTCCTCACTCATTCGCGCTCCTGTAGGCCGTCTAGAACTGGCATGGTTCAAAGAGGGGCTAGG GSTp.3 NM_000852GAGACCCTGCTGTCCCAGAACCAGGGAGGCAAGACCTTCATTGTGGGAGACCAGATCTCCTTCGCTGACTACAACCGSTT1.3 NM_000853CACCATCCCCACCCTGTCTTCCACAGCCGCCTGAAAGCCACAATGAGAATGATGCACACTGAGGCC GUS.1NM_000181CCCACTCAGTAGCCAAGTCACAATGTTTGGAAAACAGCCCGTTTACTTGAGCAAGACTGATACCACCTGCGTGHOXB13.1 NM_006361CGTGCCTTATGGTTACTTTGGAGGCGGGTACTACTCCTGCCGAGTGTCCCGGAGCTCGCTGAAACCCTGTGHSD17B1.1 NM_000413CTGGACCGCACGGACATCCACACCTTCCACCGCTTCTACCAATACCTCGCCCACAGCAAGCAAGTCTTTCGCGAGGCG HSD17B2.1 NM_002153GCTTTCCAAGTGGGGAATTAAAGTTGCTTCCATCCAACCTGGAGGCTTCCTAACAAATATCGCAGGCAHSD17B4.1 NM_000414TTGTCCTTTGGCTTTGTCACGAGAGTTGTGAGGAGAATGGTGGCTTGTTTGAGGTTGGAGCAGGATGGATTGIL17RB.2 NM_018725ACCCTCTGGTGGTAAATGGACATTTTCCTACATCGGCTTCCCTGTAGAGCTGAACACAGTCTATTTCATTGGGGCCIMMT.1 NM_006839CTGCCTATGCCAGACTCAGAGGAATCGAACAGGCTGTTCAGAGCCATGCAGTTGCTGAAGAGGAAGCCAGAAAAGCKi-67.2 NM_002417CGGACTTTGGGTGCGACTTGACGAGCGGTGGTTCGACAAGTGGCCTTGCGGGCCGGATCGTCCCAGTGGAAGAGTTGTAA LIPA.1 NM_000235CCAGTTGTCTTCCTGCAACATGGCTTGCTGGCAGATTCTAGTAACTGGGTCACAAACCTTGCCAACAGMDH2.1 NM_005918CCAACACCTTTGTTGCAGAGCTGAAGGGTTTGGATCCAGCTCGAGTCAACGTCCCTGTCATTG mGST1.2NM_020300ACGGATCTACCACACCATTGCATATTTGACACCCCTTCCCCAGCCAAATAGAGCTTTGAGTTTTTTTGTTGGATATGGA MGST3.1 NM_004528AGCTGTTGGAGGTGTTTACCACCCGCGTATAGCTTCTGGCCTGGGCTTGGCCTGGATTGTTGGACGAMMTV-like AF346816CCATACGTGCTGCTACCTGTAGATATTGGTGATGAACCATGGTTTGATGATTCTGCCATTCAAACCTTTAGGenv.3 MPV17.1 NM_002437CCAATGTGTTGCTGTTATCTGGAACTCCTACCTGTCCTGGAAGGCACATCGGCTCTAAGCCTGCCTCACTCCATMVP.1 NM_017458ACGAGAACGAGGGCATCTATGTGCAGGATGTCAAGACCGGAAAGGTGCGCGCTGTGATTGGAAGCACCTACATGCNAT1.1 NM_000662TGGTTTTGAGACCACGATGTTGGGAGGGTATGTTTACAGCACTCCAGCCAAAAAATACAGCACTGGCATGATTCANAT2.1 NM_000015TAACTGACATTCTTGAGCACCAGATCCGGGCTGTTCCCTTTGAGAACCTTAACATGCATTGTGGGCAAGCCATNCOA2.1 NM_006540AGTGACCTCCGTGCCTACGTCAGGGCTGTCCTCCATGGGTCCCGAGCAGGTTAATGATCCTGCTCTGAGGGGAGNDUFA7.1 NM_005001GCAGCTACGCTACCAGGAGATCTCCAAGCGAACTCAGCCTCCTCCCAAGCTCCCTGTGGGTCCTAGCCACAAGCTCTCC NQO1.1 NM_000903CAGCAGACGCCCGAATTCAAATCCTGGAAGGATGGAAGAAACGCCTGGAGAATATTTGGGATGAGACACCANQO2.1 NM_000904AGCGCTCCTTTCCGTAACCACGGGAGGCACGGCCGAGATGTACACGAAGACAGGAGTCAATGGA P53.2NM_000546CTTTGAACCCTTGCTTGCAATAGGTGTGCGTCAGAAGCACCCAGGACTTCCATTTGCTTTGTCCCGGGPAI1.3 NM_000602CCGCAACGTGGTTTTCTCACCCTATGGGGTGGCCTCGGTGTTGGCCATGCTCCAGCTGACAACAGGAGGAGAAACCCAGCA PR.6 NM_000926GCATCAGGCTGTCATTATGGTGTCCTTACCTGTGGGAGCTGTAAGGTCTTCTTTAAGAGGGCAATGGAAGGGCAGCACAACTACT PR.12 NM_000926GTTCCATCCCAAAGAACCTGCTATTGAGAGTAGCATTCAGAATAACGGGTGGAAATGCCAACTCCAGAGTTTCPRAME.3 NM_006115TCTCCATATCTGCCTTGCAGAGTCTCCTGCAGCACCTCATCGGGCTGAGCAATCTGACCCACGTGCPRAME.4 NM_006115CCACTGCTCCCAGCTTACAACCTTAAGCTTCTACGGGAATTCCATCTCCATATCTGCCTTGCAG PRAME NM_006115ATCAGGCACAGAGATAGAGGTGACTGGGGCCCAGGCAGTGGCAGAAGGAAGCCCGAGTTGAAAGAintron 5.1 PRDX2.1 NM_005809GGTGTCCTTCGCCAGATCACTGTTAATGATTTGCCTGTGGGACGCTCCGTGGATGAGGCTCTGCGGCTGPRDX3.1 NM_006793TGACCCCAATGGAGTCATCAAGCATTTGAGCGTCAACGATCTCCCAGTGGGCCGAAGCGTGGAAGAAACCCTCCGCTTGG PRDX4.1 NM_006406TTACCCATTTGGCCTGGATTAATACCCCTCGAAGACAAGGAGGACTTGGGCCAATAAGGATTCCACTTCTTTCAGPRDX6.1 NM_004905CTGTGAGCCAGAGGATGTCAGCTGCCAATTGTGTTTTCCTGCAGCAATTCCATAAACACATCCTGGTGTCATCACARPLPO.2 NM_001002CCATTCTATCATCAACGGGTACAAACGAGTCCTGGCCTTGTCTGTGGAGACGGATTACACCTTCCCACTTGCTGASC5DL.1 NM_006918CGCCTACATAAACCTCACCATATTTGGAAGATTCCTACTCCATTTGCAAGTCATGCTTTTCACCCTATTGATGGSOD1.1 NM_000454TGAAGAGAGGCATGTTGGAGACTTGGGCAATGTGACTGCTGACAAAGATGGTGTGGCCGATGTGTCTATTSOD2.1 NM_000636GCTTGTCCAAATCAGGATCCACTGCAAGGAACAACAGGCCTTATTCCACTGCTGGGGATTGATGTGTGGGAGCACGCT SOD3.1 NM_003102CCATAAGCCCTGAGACTCCCGCCTTTGACCTGACGATCTTCCCCCTTCCCGCCTTCAGGTTCCTCCTASRD5A2.1 NM_000348GTAGGTCTCCTGGCGTTCTGCCAGCTGGCCTGGGGATTCTGAGTGGTGTCTGCTTAGAGTTTACTCCTACCCTTCCAGGGA STK15.2 NM_003600CATCTTCCAGGAGGACCACTCTCTGTGGCACCCTGGACTACCTGCCCCCTGAAATGATTGAAGGTCGGASTK15.8 NM_003600GCCCCCTGAAATGATTGAAGGTCGGATGCATGATGAGAAGGTGGATCTCTGGAGCCTTGGA STK15 NM_003600int2CATTCACATTTATAAACCCACATGGAGGTTGGTCTTGTCGGGAATTCTTTCCGCCTTTACTTTGGATTintron 2.1 STK15  NM_003600int4GCGAGGAATGAACCCACAGACTCTTTTGCTTTTAGCGGTCTAACAGAGGCTAAGAGTCTAAATCCACTGGTTCTCATintron 4.1 GC SULT1E1.1 NM_005420ATGGTGGCTGGTCATCCAAATCCTGGATCCTTTCCAGAGTTTGTGGAGAAATTCATGCAAGGACAGGTTCCTTATSULT4A1.1 NM_014351CACCTGCCCTACCGCTTTCTGCCCTCTGACCTCCACAATGGAGACTCCAAGGTCATCTATATGGCTCGCAACCCSURV.2 NM_001168TGTTTTGATTCCCGGGCTTACCAGGTGAGAAGTGAGGGAGGAAGAAGGCAGTGTCCCTTTTGCTAGAGCTGACAGCTTTG TBP.1 NM_003194GCCCGAAACGCCGAATATAATCCCAAGCGGTTTGCTGCGGTAATCATGAGGATAAGAGAGCCACG TFRC.3NM_003234GCCAACTGCTTTCATTTGTGAGGGATCTGAACCAATACAGAGCAGACATAAAGGAAATGGGCCTGAGTTST.1 NM_003312GGAGCCGGATGCAGTAGGACTGGACTCGGGCCATATCCGTGGTGCCGTCAACATGCCTTTCATGGACTTUGT1A3.1 NM_019093GATGCCCTTGTTTGGTGATCAGATGGACAATGCAAAGCGCATGGAGACTAAGGGAGCTGGAGTGACCCTUGT2B7.2 NM_001074CAATGGCATCTACGAGGCAATCTACCATGGGATCCCTATGGTGGGGATTCCATTGTTTGCCGATCAACCTGupa.3 NM_002658GTGGATGTGCCCTGAAGGACAAGCCAGGCGTCTACACGAGAGTCTCACACTTCTTACCCTGGATCCGCAGVDAC1.1 NM_003374GCTGCGACATGGATTTCGACATTGCTGGGCCTTCCATCCGGGGTGCTCTGGTGCTAGGTTACGAGGGCTGGVDAC2.1 NM_003375ACCCACGGACAGACTTGCGCGCGTCCAATGTGTATTCCTCCATCATATGCTGACCTTGGCAAAGCT XPC.1NM_004628GATACATCGTCTGCGAGGAATTCAAAGACGTGCTCCTGACTGCCTGGGAAAATGAGCAGGCAGTCATTGAAAG

Study Methods Gene Expression

For each patient sample included in the study, 50 ng of RNA extractedfrom a FPET sample was amplified using commercially available RNAamplification kits and protocols (Genisphere). Expression levels of testand reference genes listed in Table 5 were reported as (C_(T)) valuesfrom the qRT-PCR assay (TaqMan®). Based on the relative invariability oftheir measured expression in study samples and on the lack of observedcorrelation between their measured expression and clinical outcome,CDH1, TBP, EPHX1, SERPINE1 and CD68 were chosen as reference genes. Testgene expression values were normalized relative to the mean of thesereference genes. Reference-normalized expression measurements typicallyrange from 0 to 15, where a one unit increase generally reflects a2-fold increase in RNA quantity.

Main effect Cox proportional hazard models (D. R. Cox (1972) RegressionModels and Life-Tables (with discussion). J Royal Statistical Soc. B,34:187-220) were utilized to compare the additional contribution of geneexpression beyond standard clinical prognostics variables, includingage, clinical tumor size, and tumor grade. A test for comparing thereduced model, excluding the gene expression variable, versus thecompeting full model including the gene variable of interest, called thelikelihood ratio test (Ronald Fisher (1922) “On the MathematicalFoundations of Theoretical Statistics”, Phil. Trans. Royal Soc., seriesA, 222:326, 1922; Leonard Savage (1962), The Foundations of StatisticalInference (1962)) was utilized to identify statistically significantprognostic genes.

Study Results

Using the methods described above, 34 genes were identified, for whichthe expression level was found to be significantly correlated with DRFS(p<0.1). The genes are shown in Table 8 together with Hazard Ratio andp-values. Results utilizing two distinct probe primer sets designed tomeasure distinct expression products of the PGR gene are shown. ThePR.12 probe primer set is targeted specifically toward PGR-B mRNA, whichgives rise to a longer translation product than does PGR-A mRNA. PR.6recognizes both PGR-A and PGR-B. Measurement using PR.12 resulted in alower Hazard Ratio than did PR.6, indicating that PGR-B may be the morepowerful predictor of clinical outcome.

TABLE 8 Gene LR Official Amplicon Name Hazard HR HR P- Symbol (Results)Ratio 95% LCL 95% UCL Value BCL2 Bcl2 intron 1 0.64 0.52 0.80 0.000250kb.1 GSTM2 GSTM2 gene.4 0.64 0.49 0.83 0.0003 GSTM3 GSTM3.6 0.57 0.420.78 0.0003 SCUBE2 CEGP1.6 0.76 0.65 0.88 0.0003 BCL2 Bcl2-beta.1 0.620.47 0.81 0.0007 GSTM1 GSTM1.1 0.71 0.58 0.86 0.0009 PGR PR.6 0.81 0.710.92 0.0019 MVP MVP.1 0.44 0.26 0.74 0.0026 GSTM4 GSTM4.1 0.68 0.53 0.870.0044 PGR PR.12 0.64 0.46 0.90 0.0067 BIRC5 SURV.2 1.41 1.08 1.820.0091 NAT1 NAT1.1 0.85 0.74 0.97 0.0161 CRYZ CRYZ.1 0.60 0.38 0.930.0263 GPX1 GPX1.2 0.41 0.19 0.88 0.0263 MKI67 Ki-67.2 1.41 1.02 1.930.0270 PRAME PRAME.3 1.17 1.02 1.33 0.0270 PPIH CYP.1 0.58 0.36 0.920.0283 CYP17A1 CYP17A1.1 0.69 0.49 0.99 0.0323 IL17RB IL17RB.2 0.81 0.680.98 0.0334 CAT CAT.1 0.63 0.41 0.96 0.0400 CYP4Z1 CYP4Z1.1 0.86 0.751.00 0.0416 ESR1 EstR1.1 0.87 0.77 0.99 0.0418 GPX2 GPX2.2 0.68 0.480.98 0.0419 PRDX3 PRDX3.1 0.55 0.31 0.98 0.0454 STK6 STK15.2 1.63 0.992.69 0.0475 GSTM5 GSTM5.2 0.77 0.58 1.02 0.0493 SC5DL SC5DL.1 0.65 0.421.00 0.0520 CTSL2 CTSL2.10 1.22 1.00 1.49 0.0620 VDAC1 VDAC1.1 1.89 0.963.72 0.0689 PLAU upa.3 0.66 0.43 1.03 0.0716 TFRC TFRC.3 1.49 0.97 2.300.0759 NQO1 NQO1.1 1.42 0.95 2.13 0.0803 GSTP1 GSTp.3 0.72 0.49 1.050.0840 ATP5A1 ATP5A1.1 0.56 0.30 1.07 0.0850 GUSB GUS.1 0.67 0.42 1.080.0861

Two genes from the glutathione peroxidase family, GPX1 and GPX2, werepositive prognosticators. GPX1 gave a very strong positive Cox value(H.R.=0.41, p=0.0263) and GPX2 was also strongly positive (H.R.=0.68,p=0.0419). GPX1 encodes a selenium-dependent glutathione peroxidase thatfunctions in the detoxification of hydrogen peroxide, and is one of themost important antioxidant enzymes in humans. GPX1 overexpression delayscell growth and protects from GSH and H₂O₂ toxicity. Interestingly,these biological activities are similar to BCL2, another strong positiveprognostic indicator in breast. GPX2 also encodes a selenium-dependentglutathione peroxidase and is one of two isoenzymes responsible for themajority of the glutathione-dependent hydrogen peroxide-reducingactivity in the epithelium of the gastrointestinal tract. Studies inknockout mice indicate that mRNA expression levels respond to luminalmicroflora, suggesting a role of GPX2 in preventing inflammation in theGI tract,

Another strong positive Cox value was found with peroxiredoxin 3,(PRDX3; H.R.=0.55, p=0.0454). This gene encodes a protein withantioxidant function and is localized in the mitochondrion. PRDX3 is amember of a gene family that is responsible for regulation of cellularproliferation, differentiation, and antioxidant functions.

The strong positive prognostic effect of CRYZ (H.R.=0.60, p=0.0263) isalso consistent with its function as an antioxidant. CRYZ encodes themajor detoxifying enzyme quinone reductase (QR) [NAD(P)H:quinoneoxidoreductase]. It is hypothesized that QR inhibits estrogen-inducedDNA damage by detoxification of reactive catecholestrogens. CRYZ istranscriptionally activated by anti-estrogen liganded Eβ. Up-regulationof QR, either by overexpression or induction by tamoxifen, can protectbreast cells against oxidative DNA damage caused by estrogenmetabolites, representing a possible novel mechanism of tamoxifenprevention against breast cancer. (See Table 9 Univariate Cox PHregression analysis. Assays are ordered by p-value, with p-values≦0.05considered significant. Specimens from 125 breast cancer patients wereassayed.)

TABLE 9 Hazard HR HR Univariate Analysis Ratio 95% LCL 95% UCL P-ValueCRYZ.1 0.60 0.38 0.93 0.0263 CYP1B1.3 0.81 0.55 1.19 0.2852 UGT2B7.21.07 0.94 1.22 0.3763 SULT1E1.1 1.08 0.91 1.28 0.3862 COMT.1 0.87 0.421.81 0.711 SULT4A1.1 1.01 0.82 1.25 0.9427 CYP1A1.2 1.01 0.82 1.23 0.949UGT1A3.1 1.00 0.78 1.27 0.974

The cytochrome P450 proteins are monooxygenases which catalyze manyreactions involved in drug metabolism and synthesis of cholesterol,steroids and other lipids. Two of the five cytochrome P450 superfamilymembers tested were also significant indicators of positive prognosis.CYP17A1 (H.R.=0.69, p=0.0323) localizes to the endoplasmic reticulum. Ithas both 17alpha-hydroxylase and 17,20-lyase activities and is a keyenzyme in the steroidogenic pathway that produces progestins,mineralocorticoids, glucocorticoids, androgens, and estrogens. Therecently discovered CYP4Z1 (H.R.=0.86, p=0.0416), also an endoplasmicreticulum integral membrane protein, is restricted to expression inbreast and showed a clear over-expression in 52% of breast cancersamples in one study.

The antioxidant protein catalase (CAT) is located at the peroxisome andscavenges H₂O₂. Consistent with its function was the finding that CATexpression correlated with positive prognosis (H.R.=0.63, p=0.040).

The sterol-C5-desaturase like gene (SC5DL) encodes an enzyme that isinvolved in cholesterol biosynthesis. Expression of SC5DL isdownregulated in human ovarian carcinomas in vivo during Taxol(R)(paclitaxel) treatment. In our study, increased expression of SC5DL wasa positive prognostic indicator (H.R.=0.65, p=0.052).

NAT1, a xenobiotic-metabolizing enzyme, is an ERα-responsive gene inhuman breast cancer and has been suggested as a candidate molecularpredictor of antiestrogen responsiveness. In a 97 ERalpha-positivebreast tumor study, relapse-free survival was longer among patients withNAT1-overexpressing tumors (P=0.000052), and retained prognosticsignificance in Cox multivariate regression analysis (P=0.0013). In ourcurrent study, we show that NAT1 maintains a positive prognosticsignificance in a univariate Cox model (H.R.=0.85, p=0.0161) NAT1 alsoshows a strong expression correlation with ER (R=0.67), consistent withit being an ERα responsive gene.

The glutathione S-transferase pi gene (GSTP1) is a polymorphic geneencoding active, functionally different GSTP1 variant proteins that arethought to function in xenobiotic metabolism and play a role insusceptibility to cancer. GSTp was a positive prognostic indicator inour study (H.R.=0.72, p=0.084).

One skilled in the art will recognize numerous methods and materialssimilar or equivalent to those described herein, which could be used inthe practice of the present invention. Indeed, the present invention isin no way limited to the methods and materials described. While thepresent invention has been described with reference to what areconsidered to be the specific embodiments, it is to be understood thatthe invention is not limited to such embodiments. To the contrary, theinvention is intended to cover various modifications and equivalentsincluded within the spirit and scope of the appended claims. Forexample, while the disclosure is illustrated by identifying genes andgroups of genes useful in determining prognosis for patients diagnosedwith invasive breast cancer, similar methods in determining prognosisfor patients diagnosed with cancer of other cell types, includingprostate and ovarian cancer.

1.-38. (canceled)
 39. A method of predicting the likelihood of a positive clinical outcome for a human subject diagnosed with breast cancer, comprising: assaying an expression level of an RNA transcript of VDAC1 in a tumor sample obtained from said subject; normalizing the RNA level of VDAC1 against the RNA level of one or more reference genes to obtain a normalized expression level of VDAC1; using the normalized expression level of VDAC1 to generate information comprising a prediction of the clinical outcome for said subject, wherein the normalized expression level of VDAC1 is positively correlated with a positive clinical outcome.
 40. The method of claim 39, wherein said expression level is obtained by a method of gene expression profiling.
 41. The method of claim 39, wherein said method is a polymerase chain reaction-based method.
 42. The method of claim 39, wherein the tumor sample is obtained by biopsy.
 43. The method of claim 39, wherein the tumor sample comprises fragmented RNA.
 44. The method of claim 39, further comprising generating a report based on the information.
 45. The method of claim 44, wherein the report comprises information concerning a risk of cancer recurrence for said subject.
 46. The method of claim 44, wherein the report further comprises information to guide a cancer treatment decision for said subject.
 47. The method of claim 39, further comprising assaying an expression level of at least one additional RNA transcript selected from the group GSTM1, GSTM2, GSTM3, GSTM4, GSTM5, TFRC, MVP, PRAME, PPIH, NAT1, and CYP4Z1.
 48. The method of claim 39, wherein said clinical outcome is expressed in terms of Recurrence-Free Interval, Overall Survival, Disease-Free Survival, or Distant Recurrence-Free Interval. 